KR20090130382A - PDGFRbeta-SPECIFIC INHIBITORS - Google Patents

Abstract

The invention is directed to novel PDGFRF-specific antagonists. The antagonists include antibodies, which can be bispecific. The antibodies are used to reduce or inhibit tumor growth and or to treat an angiogenic disease. The invention also includes combinations of PDGFRF-specific antagonists with VEGFR antagonists for such treatments. The antagonists can further be administered in combination with other anti-angiogenic or anti-neoplastic drugs.

Description

PDVFRβ-specific inhibitors {PDGFRβ-SPECIFIC INHIBITORS}

<Cross reference to related application>

This application claims priority based on US patent provisional application 60 / 923,979, filed April 17, 2007, the disclosure of which is incorporated herein by reference.

The present invention relates to methods and compositions for inhibiting angiogenesis and tumor growth. The present invention demonstrates the effect of specific inhibition of platelet derived growth factor receptor-beta (PDGFRβ) on angiogenesis and provides for the treatment of angiogenesis using PDGFRβ specific antagonists alone or in combination with inhibitors of VEGF receptors. do. Specific inhibitors may be antibodies or antigen binding fragments thereof. In addition, the inhibitor may be bispecific.

Platelet derived growth factor (PDGF) is a family of potent mitogens for almost all mesenchymal-derived cells. There are four PDGF isotypes, namely A, B, C and D, and they have five different disulfide-linked dimer proteins, PDGF-AA, -BB, -AB, -CC, and -DD. Form. These growth factors exert their cellular effects through two structurally related tyrosine kinase receptors, PDGF receptor α (PDGFRα) and PDGF receptor β (PDGFRβ) (Sandy, JR, 1998, Br. J. Orthod. 25 : 269-74] (Betsholtz, C. et al., 2001, Bioessays 23: 494-507).

PDGFRα and PDGFRβ are structurally similar and can form heterodimers as well as homodimers. PDGF-BB and PDGF-DD are the major activators of ββ homodimeric receptors. PDGF-AA activates only αα receptor dimers, while PDGF-AB, PDGF-BB and PDGF-CC activate αα and αβ receptor dimers. Dimer ligand molecules bind to two receptor proteins simultaneously and induce receptor dimerization, autophosphorylation of specific residues within the cytoplasmic domain of the receptor, and intracellular signaling. Ultimately, activation of the PDGFRβ signaling pathway induces a variety of cellular responses, including cell proliferation and migration.

Angiogenesis is thought to be essential for both tumor growth and metastasis. The development, or angiogenesis, of the vascular system requires the development of two major cell types, namely endothelial cells (EC) and perivascular / vascular smooth muscle cells (SMC), into mature blood vessels. The involvement of the PDGF-B / PDGFRβ signaling pathway in angiogenesis is proposed by PDGF-B and PDGFRβ knockout mice. The PDGF-B and PDGFRβ knockout phenotypes are substantially identical in that mice show bleeding due to a loss of coverage of perivascular and smooth muscle cells in blood vessels. Studies show that PDGFRβ is involved in the recruitment of perivascular cells to capillaries and the development of smooth muscle cells in blood vessels. Mobilization of perivascular cells to coat the generator vessels is essential for stabilization and further establishment of the vascular network. Veins without adequate perivascular cells are more susceptible to VEGF inhibition than well established mature vessels.

Many tyrosine kinase inhibitors being developed as antitumor agents have been shown to inhibit PDGFRβ. However, these compounds have a number of tyrosine kinase targets. For example, imatinib mesylate (Gleevec® / ST571) was developed as an Abelson (Abl) tyrosine kinase inhibitor and also inhibits c-kit, PDGFRα and PDGFRβ. Sunitinib malate (Sutent® / SU 11248) is a wide range of orally available multitargeted tyrosine kinase inhibitors with activity against VEGFR, PDGFR, c-KIT and FLT-3. CP-673,451 is an inhibitor of both PDGFRα and PDGFRβ. Since these small molecule antagonists are not specific for these receptors, administration of these compounds may be due to the contribution of PDGFRβ signaling to angiogenesis, including tumor associated angiogenesis, tumor stimulation and growth, or unnecessary targeting of multiple receptors. It is not possible to distinguish the toxicity associated with.

Accordingly, the present invention provides PDGFRβ-specific antagonists, demonstrates the role of PDGFRβ in angiogenesis, including angiogenesis that supports tumor growth and survival, and provides methods for treating tumor and angiogenic diseases. The present invention further demonstrates the benefits of simultaneously inhibiting signaling through PDGFRβ and VEGFR.

<Overview of invention>

The present invention provides PDGFRβ-specific antagonists of PDGFRβ mediated signaling. In an embodiment of the invention, the PDGFRβ-specific antagonist is an antibody that specifically binds platelet derived growth factor receptor-β (PDGFRβ). One of said antibodies for SEQ ID NO: 20 in CDRH1; For SEQ ID NO: 22 in CDRH2; For SEQ ID NO: 24 in CDRH3; For SEQ ID NO: 28 in CDRL1; For SEQ ID NO: 30 in CDRL2; And a complementarity determining region at least about 90% homologous or about 90% identical to SEQ ID NO: 32 in CDRL3. Another such antibody comprises the heavy chain variable domain amino acid sequence of SEQ ID NO: 18 and the light chain variable domain amino acid sequence of SEQ ID NO: 26. Another embodiment of the antibody is directed to SEQ ID NO: 4 in CDRH1; For SEQ ID NO: 6 in CDRH2; For SEQ ID NO: 8 in CDRH3; For SEQ ID NO: 12 in CDRL1; For SEQ ID NO: 14 in CDRL2; And a complementarity determining region at least about 90% homologous or about 90% identical to SEQ ID NO: 16 in CDRL3. Another such antibody comprises the heavy chain variable domain amino acid sequence of SEQ ID NO: 2 and the light chain variable domain amino acid sequence of SEQ ID NO: 10. The present invention further includes PDGFRβ-specific antibodies that bind to epitopes that are identical or overlapping with one of the aforementioned antibodies.

Antibodies of the invention can be chimera, humanized or human antibodies. Antibodies of the invention can also be antigen binding fragments such as single chain antibodies, Fab and F (ab ') ₂ fragments, single chain Fv, and single domain antibodies. Antibodies also include diabodies and triabodies. In addition, the antibody may be bispecific.

The invention further provides isolated polynucleotides encoding the antibodies, expression vectors, and host cells producing the antibodies.

The present invention provides a method of modulating the activity of PDGFRβ in a mammal comprising administering to the mammal an effective amount of platelet derived growth factor receptor-β (PDGFRβ) -specific antagonist. In one embodiment of the invention, an effective amount of PDGFRβ-specific antagonist is used to inhibit angiogenesis in a mammal. In another embodiment, an effective amount of PDGFRβ-specific antagonist is used to reduce tumor growth in a mammal. The PDGFRβ-specific antagonist may be an antibody that binds PDGFRβ, or any other substance that binds to PDGFRβ or PDGFRβ ligands and reduces or blocks PDGFRβ-mediated signaling. The method of treatment may further comprise a combination administration of VEGFR antagonists. Alternatively, the method of treatment may use a substance, for example a bispecific antibody, which is a PDGFRβ-specific antagonist and at the same time a VEGFR antagonist.

According to the present invention, the treatment of tumors and angiogenic conditions may further comprise the administration of anti-neoplastic agents such as chemotherapy or radiation.

1 shows quantitative receptor binding and blocking assays for 1B3, an anti-PDGFRβ antibody. (A) Purified Fab fragment or full length 1B3 IgG was added to mPDGFRβ / Fc-coated plates (1 μg / ml) and incubated for 1 h at RT. Plate-bound antibodies were detected using goat anti-human-κ antibody-HRP. (B) 1B3 antibody or Fab was first mixed with fixed amount of mPDGFRβ / Fc (50 ng) and incubated for 30 minutes at RT. The mixture was then transferred to a plate precoated with PDGF-BB (0.5 μg / ml) and incubated for 1 h at RT. Plate-bound mPDGFRβ / Fc was detected using goat anti-human Fc antibody-HRP conjugate. IMC-1121 is an antibody produced against human VEGFR2. Data represent mean ± SD of triplicate samples.

FIG. 2 depicts the inhibition of PDGF-BB-induced receptor phosphorylation and downstream signaling molecules MAPK and Akt caused by binding of antibody 1B3 to cell surface PDGFRβ. (A) NIH / 3T3 (fibroblasts), D122 (Lewis lung carcinoma), 4T1 (breast cancer), B16 (melanoma), incubated with anti-PDGFRβ antibody 1B3 and anti-human Fc antibody-FITC conjugate ) And FACS analysis of H5V (PmT-transformed endothelial cells) cells. (B) Inhibition of PDGF-BB-induced receptor phosphorylation in NIH-3T3 cells. (C) Inhibition of PDGF-BB-induced receptor phosphorylation in D122 cells. (D) Inhibition of PDGF-BB-stimulated phosphorylation of AKT and p44 / 42 MAP kinases in D122 cells.

3 shows the effect of treatment with anti-mPDGFRβ antibody 1B3 on the growth of human xenograft tumors in vivo. In athymic nude mice using six xenograft models, SK-OV-3, OV-CAR-8, BxPC-3, OV-CAR-5, Caki-1 and NCI-H460 (A to F, respectively) The effect of 1B3 on xenograft tumors was examined. Mice with established tumors (about 250-300 mm ³ ) were randomly sorted and twice weekly (except for the BxPC-3 model treated with mice three times weekly) 1B3, normal humans by intraperitoneal injection at the indicated dose. IgG, or saline. Tumor volume is measured twice weekly and presented as mean ± SEM. ^* 1 represents the loss of one mouse due to severe illness. 1B3 binds to mouse PDGFRβ but not to human PDGFRβ. Thus, the response observed for treatment is due to the inhibition of stimulation of the host cell.

4 shows the effect of combination treatment with anti-mPDGFRβ antibody 1B3 and anti-mVEGFR2 antibody DC101 on the growth of human xenograft tumors in vivo. Subcutaneous xenografts of BxPC-3 (A) or 5 × 10 ⁶ NCI-H460 (B) were established in female athymic (nu / nu) mice. When tumors reached about 300-350 mm ³ , mice were randomized and divided into 4 groups (n = 12), USP saline, 40 mg / kg 1B3, 40 mg / kg DC101 or 40 mg / kg 1B3 + 40 mg / kg DC101 three times weekly. Tumor volume is measured twice weekly and presented as mean ± SEM. ^* 1 represents the loss of one mouse due to severe illness. 1B3 binds to mouse PDGFRβ but not to human PDGFRβ. DC101 binds to mouse VEGFR2 but not to human VEGFR2. Thus, the response observed for treatment is due to the inhibition of stimulation of the host cell.

Figure 5 shows the results of immunohistochemical analysis of human xenografts after treatment with 1B3, DC101, or DC101 + 1B3. At 34 days post-treatment (BxPC3 model, panels A to C) or 50 days (NCI-H460 model, panels D to F), 6 mice from each group were sacrificed and tumors were processed for IHC analysis. Tumors were sectioned and double stained for both blood vessels (anti-CD31) and perivascular / SMC coverage (α-SMA, panels B and E). Five digital immunofluorescence images of the tumor periphery and tumor center were obtained from each section, for total CD31 ⁺ vessels (Panels A and D), α-SMA ⁺ vessels (Panels B and E), and for total CD31 ⁺ vessels. Dual were quantified for percentage of CD31 ⁺ / α-SMA ⁺ (Panels C and F). Two different quantitative methods were used to measure the vascular distribution of individual tumor sections: vessel density (number of vessels / mm ³ ) and percentage of vessel area (% vessel area to total tumor area). For statistical analysis, a two-way ANOVA followed by Fisher LSD method was used for multiple comparisons (Sigma Stat 3.1 Systat Software, Inc., Point Richmond, CA, USA). ). * P <0.05 compared to the brine group. Empty rods, tumor periphery; Solid rod, center of tumor.

6 depicts receptor binding and blocking of ligand (PDGF-BB) to human PDGFRβ and murine PDGFRβ by PDGFRβ-specific human antibody 2C5. Binding and ligand blocking are compared to PDGFRβ-specific antibody 1B3 which only binds to murine PDGFRβ.

FIG. 7 is a Western blot demonstrating the inhibition of ligand (PDGF-BB) induced phosphorylation of PDGFRβ in human Caki-1 tumor cells by anti-PDGFRβ antibody 2C5. The antibody also inhibits phosphorylation of Akt and P44 / 42.

FIG. 8 shows Western blot demonstrating inhibition of ligand (PDGF-BB) induced phosphorylation of PDGFRβ in mouse NIH-3T3 and D122 cell lines by anti-PDGFRβ antibody 2C5.

FIG. 9 depicts the growth of six subcutaneous human xenograft tumors in nude mice treated with anti-PDGFRβ antibody 1B3 (specific to murine PDGFRβ) and / or 2C5 (binding to both human and murine PDGFRβ).

10 depicts the growth of six subcutaneous human xenograft tumors in nude mice treated with anti-PDGFRβ antibody 2C5 (which binds to both human and murine PDGFRβ) and murine VEGFR-2 specific antibodies (DC101).

11 shows the results of a cell based migration assay demonstrating that 2C5 inhibits PDGF-BB-stimulated cell migration.

12 shows antitumor activity of 2C5 / chemotherapy combination and 2C5 / chemotherapy / DC101 therapy in tumor xenograft models.

Figure 13 shows antitumor activity of 2C5 / 3G3 and 2C5 / 1E10 combination therapy in tumor xenograft models.

14 shows the effect of 2C5 on PDGFRβ, PDGF-BB, VEGF and FGF expression.

15 shows the binding of 2C5 to the PDGFRβ domain.

Figure 16 shows the DNA and amino acid sequences of the variable heavy and variable light chains of 1B3 underlined CDRs.

Figure 17 shows the DNA and amino acid sequences of the variable heavy and variable light chains of 2C5 underlined CDRs.

The present invention provides antibodies or fragments thereof that are specific for PDGFRβ. Antibodies of the invention include those that specifically bind to domains 1 and 2 of PDGFRβ. Antibodies of the invention include 2C5 and 1B3.

The present invention also provides compositions and methods for the treatment of tumors and angiogenic diseases using the antibodies described above and using anti-PDGFRβ antagonists in combination with VEGFR antagonists. In particular, the present invention provides specific targeting of the PDGF-B / PDGFRβ signaling pathway for the treatment of cancer by anti-tumor and anti-angiogenic mechanisms. The present invention also provides for the treatment of angiogenic diseases.

Accordingly, the present invention provides platelet derived growth factor receptor-β (PDGFRβ) antagonists specific for PDGFRβ. PDGFRβ-specific antagonists are biological molecules that preferentially inhibit PDGFRβ mediated signaling. According to the present invention, PDGFRβ-specific antagonists mediate the inhibition of PDGFRβ mediated signaling at least 3x, or at least 5x, or at least 10x greater than the inhibition of PDGFRα. One method of determining PDGFRβ-specific inhibition is the inhibition of PDGF-induced activation of cells engineered to express PDGFRβ but not to express PDGFRα, PDGF of cells engineered to express PDGFRα but not to express PDGFRβ. It is compared with the inhibition of induced activation. Another method compares the inhibition of PDGF-induced activation using PDGF (eg PDGF-DD) that preferentially activates PDGFRβ and PDGF (eg PDGF-AA) that preferentially activates PDGFRα. will be. In an embodiment of the invention, the PDGFRβ-specific antagonist is an antibody that binds PDGFRβ. In another embodiment of the invention, the PDGFRβ-specific antagonist is a small molecule.

Anti-angiogenic methods that target VEGF receptors are increasingly recognized as effective for anti-tumor therapy. For example, anti-VEGF antibodies have been approved for the treatment of certain tumors. In addition, anti-mouse VEGFR2 antibodies blocked VEGF / VEGFR2 interactions in murine xenograft models and inhibited VEGF-stimulated proliferation and survival of vascular endothelial cells. However, although treatment with anti-VEGFR2 antibodies significantly delayed the growth of various tumors implanted in mice, tumor regression was rare. In addition, evidence accumulates suggesting that during long-term treatment some tumors may become less sensitive (or even resistant) to VEGFR2 blockade.

According to the present invention, PDGFRβ-specific inhibition significantly enhances both anti-angiogenic and anti-tumor activity of VEGFR antagonists. Accordingly, the present invention provides an improved method of inhibiting angiogenesis and reducing or inhibiting tumor growth by administering PDGFRβ-specific antagonists and optionally VEGFR antagonists. Antagonists are molecules that block, modulate, or interfere with signaling mediated by receptor tyrosine kinases (ie, PDGFRβ or VEGFR), and are antibodies, small molecules, proteins, polypeptides, ligand mimetic, antisense oligodeoxynucleotides, antisense RNA, small inhibitory RNA, triple helix forming nucleic acids, dominant negative mutants, and soluble receptor expression. In addition, PDGFRβ-specific antagonists and VEGFR antagonists can be combined into a single compound, such as but not limited to bispecific antibodies.

PDGFRβ and VEGF receptors are classified as kinase insert domain containing receptors of the same family. However, the VEGF receptor, unlike the five in PDGFRα and PDGFRβ, has seven immunoglobulin-like loops in its extracellular domain. In addition, the VEGF receptor has a longer kinase insert. The amino acid sequences of PDGFRβ and VEGFR of humans and other mammals and the nucleic acid sequences encoding them are well known in the art. Non-limiting examples of human PDGFRβ sequences are provided by GenBank Accession No. NM_002609. The nucleotide sequence encodes a protein having a cleavable signal sequence. The mature protein comprises an extracellular portion containing about 499 amino acids, a transmembrane region of 23 amino acids, and an intracellular portion of about 552 amino acids. Non-limiting examples of VEGFR sequences include NM_002019 (human VEGFR1 / Flt1), NM_002253 (human VEGFR2 / KDR / Flk1), and NM_182925 (human VEGFR3 / Flt4). The intracellular regions, including the Ig-like domain structure, transmembrane domain position, and tyrosine kinase domain of the extracellular domain of each of these receptors are well known in the art.

Antibodies of the invention bind to PDGFRβ. As used herein, "antibody" refers to an immunoglobulin (Ig) molecule and an immunologically active portion or variant of an immunoglobulin molecule. The antibody contains one or more antigen binding sites that specifically bind to the antigen. Antibodies include, but are not limited to, polyclonal, monoclonal, chimeric, and humanized antibodies. Immunologically active moieties include monovalent and divalent fragments such as Fv, single chain Fv (scFv), single variable domain (sVD), Fab, Fab 'and F (ab') ₂ fragments. Immunologically active moieties can be introduced into multivalent forms such as diabodies, triabodies and the like. The antibody further comprises an antigen binding fragment displayed on phage, and an antibody conjugate.

An “isolated antibody” has been (1) partially, substantially or completely purified from a mixture of components; (2) identified and separated from components of its natural environment and / or recovered; (3) monoclonal; (4) no other protein from the same species is present; (5) expressed by cells from different species; (6) It is an antibody which does not arise in nature. Contaminant components in its natural environment are substances that inhibit diagnostic or therapeutic uses for antibodies and may include enzymes, hormones, and other proteinaceous or nonproteinaceous solutes. Examples of isolated antibodies include affinity purified anti-PDGFRβ antibodies using PDGFRβ, anti-PDGFRβ antibodies prepared by hybridomas or other cell lines in vitro, human anti-PDGFRβ isolated from libraries such as phage libraries, And human anti-PDGFRβ antibodies derived from transgenic mice.

Generally, naturally occurring antibody molecules consist of two identical heavy chains and two light chains. Each light chain is usually covalently linked to the heavy chain by interchain disulfide bonds, and the two heavy chains are further linked to each other by a number of disulfide bonds in the hinge region. Individual chains are folded into domains of similar size (about 110-125 amino acids) and structure but with different functions. The light chain comprises one variable domain (V _L ) and one constant domain (C _L ). The heavy chain comprises one variable domain (V _H ) and three or four constant domains (C _H 1, C _H 2, C _H 3 and C _H 4) depending on the class or isotype of the antibody. In mice and humans, the isotypes are IgA, IgD, IgE, IgG and IgM, and IgA and IgG are further classified into subclasses or subtypes. The portion of the antibody consisting of the V _L and V _H domains is named “Fv” and constitutes the antigen-binding site. Single chain Fv (scFv) is an engineered protein containing a V _L domain and a V _H domain on one polypeptide chain, wherein the N terminus of one domain and the C terminus of another domain are linked by a flexible linker . "Fab" refers to the portion of an antibody consisting of V _L -C _L (ie, light chain) and V _H -C _H 1 (also named "Fd").

Antibodies of the invention include antigen binding proteins comprising a single variable domain (sVD) and sVD. The sVD binding site can be obtained from the antigen specific Fv region (including both V _H and V _L domains). Often, the binding affinity and specificity of the Fv region may appear to be primarily indicated by one of the variable domains. Alternatively, scFv can be obtained directly. Direct sources of sVD include mammals (eg, camelids) that naturally express antibodies containing only the V _H domain. In addition, phage display libraries can be prepared to express only a single variable domain. For example, human domain antibody phage display libraries are commercially available from Domantis (Cambridge, UK).

Antibody variable domains exhibit significant amino acid sequence variability, particularly from one antibody to the next at the location of the antigen binding site. Three zones called “complementarity determining zones” (CDRs) are found in each of V _L and V _H. CDRs of antibodies are often referred to as "hypervariable regions".

"Fc" is the name for a portion of an antibody that includes a paired heavy chain constant domain. In IgG ₁ antibodies, for example, Fc comprises C _H 2 and C _H 3 domains. The Fc of an IgA or IgM antibody further comprises a C _H 4 domain. Fc is associated with activation of Fc receptor binding, complement-mediated cytotoxicity and antibody-dependent cytotoxicity. In the case of natural antibodies such as IgA and IgM, which are complexes of many IgG-like proteins, complex formation requires an Fc constant domain.

Finally, the “hinge” region separates the Fab and Fc portions of the antibody, providing mobility of the Fab to each other and to the Fc, and includes multiple disulfide bonds for covalent linkage of the two heavy chains. Thus, the antibodies of the present invention are naturally occurring antibodies, bivalent fragments such as (Fab ') ₂ , monovalent fragments such as Fab, single chain antibodies, single chain Fv (scFv) that specifically bind to the antigen. , Single domain antibodies, multivalent single chain antibodies, diabodies, triabodies, and the like. In addition, antibody fragments include polypeptides having an amino acid sequence substantially similar to the amino acid sequence of the variable or hypervariable region of an antibody of the invention. Substantially identical amino acid sequences are described herein in Pearson and Lipman, Proc. Natl. Acad. Sci. USA 55: 2444-2448 (1988), at least 70%, at least about 80%, at least about 90%, at least about 95%, or at least about 99% homology with the amino acid sequences compared when determined by the FASTA search method according to Or as sequences having identity.

The present invention includes "chimeric" antibodies. The antibody generally comprises a constant domain of an antibody that is different from the variable domain of one antibody. Typically, in order to enhance the host response to the antibody target by minimizing the host immune response to the antibody and retaining the antibody effector function, the constant domain of the chimeric antibody is derived from the same species to which the chimeric antibody will be administered.

The invention also encompasses "humanized" antibodies. Humanized variable domains have been prepared in which an amino acid sequence comprising at least one complementarity determining region (CDR) of non-human origin is grafted to a human framework region (FR) (see, eg, Jones, PT et al. al., 1996, Nature 321, 522-25; Riechman, L. et al., 1988, Nature 332, 323-27; and US Pat. No. 5,530,101 (Queen et al.). Humanized constructs are particularly useful for the removal of deleterious immunogenic properties, for example where antigen binding domains from non-human sources are required to be used for the treatment of humans. Variable domains have a high degree of structural homology, which facilitates the identification of amino acid residues within variable domains corresponding to CDRs and FRs (eg, Kabat, EA, et al., 1991, Sequences of Proteins of Immunological Interest.5th ed.National Center for Biotechnology Information, National Institutes of Health, Bethesda, MD]. Thus, amino acids that are likely to participate directly in antigen binding are readily identified. In addition, methods have been developed to preserve or enhance the affinity for an antigen of a humanized binding domain comprising grafted CDRs. One method is to include a foreign framework residue in the receptor variable domain that affects the conformation of the CDR regions. The second method is to graf the foreign CDRs onto human variable domains with homology closest to the foreign variable regions (Queen, C. et al., 1989, Proc. Natl. Acad. Sci. USA 86, 10029-33 ). CDRs are most easily grafted onto different FRs by first amplifying the individual FR sequences using overlapping primers containing the desired CDR sequences and linking the resulting gene segments in subsequent amplification reactions. Grafting of CDRs onto different variable domains may further involve substitution of amino acid residues that have been filled for CDRs within folded variable domain constructs that are adjacent to or affect the conformation of the CDRs within the amino acid sequence. Thus, the humanized variable domains of the present invention include human domains comprising one or more non-human CDRs as well as those domains in which further substitutions or replacements have been made to preserve or enhance binding properties.

Antibodies of the invention can also use variable domains that become less immunogenic by replacing surface-exposed residues to make the antibody appear to itself in the immune system (Padlan, EA, 1991, Mol. Immunol. 28, 489-98 ). Antibodies can be modified by this procedure without loss of affinity (Roguska et al., 1994, Proc. Natl. Acad. Sci. USA 91, 969-973). Affinity is preserved because the internal filling of amino acid residues remains unchanged near the antigen binding site. Substitution of surface-exposed residues according to the invention for immunogenicity reduction does not mean substitution of CDR residues or adjacent residues that affect binding properties.

It is often desirable to use variable domains that are human in nature. Human antibodies include human V _H and V _L framework regions (FW) as well as human complementarity determining regions (CDR). Preferably, the entire V _H and V _L variable domains are human domains or are derived from human sequences. Antibodies can be obtained directly from human cells, for example by producing human hybridomas.

Alternatively, human antibodies can be obtained from transgenic animals into which unrearranged human Ig gene segments have been introduced and in which the endogenous mouse Ig gene has been inactivated (Brueggemann and Taussig, 1997, Curr. Opin. Biotechnol. 8, 455 -58]. Preferred transgenic animals contain very large contiguous Ig gene fragments exceeding 1 Mb in size (Mendez et al., 1997, Nature Genet. 15, 146-56), but human apes with moderate affinity are smaller genes. From transgenic animals containing locus (eg, Wagner et al., 1994, Eur. J. Immunol. 42, 2672-81); Green et al., 1994, Nature Genet. 7, 13-21).

Human antibodies can also be obtained from a library of antibody V _H and / or V _L domains. For example, variable domain libraries can be obtained from human genomic sequences or from peripheral blood lymphocytes that productively express rearranged variable region genes. In addition, the human gene library can be synthetic. In one embodiment, a variable domain library can be generated that comprises a human framework region having one or more CDRs synthesized to include random or partial random sequences. For example, a human V _H variable domain library can be generated in which members are encoded by the synthetic sequences for the human V _H gene segment and the CDR3H region (ie, the synthetic D _H -J _H gene segment). Likewise, human V _L variable domains can be encoded by synthetic sequences (ie, synthetic J _L gene segments) for human V _L gene segments and CDR3L regions. In another embodiment, the human framework may be synthetic in that it has a consensus sequence derived from a known human antibody sequence or subgroup of human sequences. In another alternative, one or more CDRs are obtained by amplification from human lymphocytes expressing the rearranged variable domains and then recombined into a particular human framework.

To screen libraries of variable domains, it is common to use phage display libraries in which a combination of human heavy and light chain variable domains is displayed on the surface of filamentous phage (see, eg, McCafferty et al., 1990 Nature 348, 552-54; see Aujame et al., 1997, Human Antibodies 8, 155-68. Combinations of variable domains are typically displayed on filamentous phage in the form of Fabs or scFvs. Libraries are screened for phage retaining combinations of variable domains with the desired antigen binding properties. Preferred single domain and variable domain combinations exhibit high affinity for the selected antigen and have little cross-reactivity to other related antigens. By screening very large repertoires of antibody fragments (eg, Griffiths et al., 1994, EMBO J. 13, 3245-60), a high variety of high affinity binding domains are isolated and many are required for antigens. It is expected to have an affinity of less than nanomolar.

In physiological immune responses, mutations and selection of expressed antibody genes can produce antibodies with high affinity for their target antigens. The V _H and V _L domains included in the antibodies of the invention can similarly be applied to in vitro or in vivo mutation and screening procedures to modify affinity and / or specificity. Thus, the binding domains of the present invention include those in which binding properties are improved by direct mutation, methods of affinity maturation, or by mutating the CDR and / or FW regions by chain shuffling. It is understood that amino acid residues, which are the major determinants of binding of a single domain antibody, may be present in the CDRs defined by Kabat, but may also include other residues. In the case of sVD, residues important for antigen binding may also include amino acids, which may potentially be located at the interface of the V _H -V _L heterodimer. Typically, phage display is used to screen such mutants to identify mutants with the required binding properties (eg, Yang et al., J. Mol Biol., 254: 392-403 ( 1995)]. Mutations can be induced in a variety of ways. One method is to randomize individual residues or combinations of residues so that, within a population of identical sequences, all twenty amino acids or a subset thereof is found at a particular position. Alternatively, mutations can be induced over a range of CDR residues by error prone PCR methods (eg, Hawkins et al., J. Mol. Biol, 226: 889-896 (1992). ] Reference). For example, phage display vectors containing heavy and light chain variable region genes may be obtained. It can be propagated in mutant strains of E. coli (see, eg, Low et al., J. Mol. Biol, 250: 359-368 (1996)). These mutagenesis methods are examples of many methods known to those skilled in the art.

The invention also includes antigen binding proteins engineered from non-immunoglobulin scaffolds. For example, S. Affibodies derived from the immunoglobulin-binding domain of S. aureus protein A do not have disulfide bonds and exhibit reversible folding. Another example is fibronectin, which has an antibody-like structure and shows a CDR-like loop. In contrast to antibodies, the fibronectin domain structure does not depend on disulfide bonds and furthermore shows high thermodynamic stability. Binding sites can be engineered into such scaffolds, for example, by varying codons at specific locations and screening for binding to the desired antigen. Codons can be randomized within a loop, flat surface, cavity, or a combination of these locations. In addition, peptide sequences can generally be inserted into a loop. Target-binding variants of the resulting library can be isolated using a selection of screening techniques known in the art and not limited to phage display, ribosomal display, bacterial or yeast surface display, and the like.

For antigen binding proteins for therapeutic purposes, various methods are available to minimize potential immunogenicity. Human scaffolds can be used and immunogenicity can be minimized, eg, by PEGylation or T-cell epitope engineering (ie, by minimizing T-cell reactive sequences).

Antigen-binding proteins from non-immunoglobulin scaffolds can often be produced more economically than immunoglobulin-type proteins. For example, the absence of disulfide bonds or free cysteine allows expression of functional molecules in the reducing environment of bacterial cytoplasm, which generally provides higher yields than peripheral cytoplasmic expression and is easier than in vitro refolding. See Binz, H.K. et al., Nat. Biotech. 23: 1257-68, 2005 disclose various such antigen-specific binding proteins and techniques for their development.

As noted above, bispecific antibodies can be provided as an alternative to combination administration. There are a variety of bispecific antibodies designed to include a variety of desirable properties. For example, bispecific diabodies have a minimum size. Bispecific antibodies with four antigen binding sites (two for each binding specificity) have binding capacity to each target similar to the corresponding native antibody. Certain bispecific antibodies comprise an Fc region and thus retain the effector functions of natural antibodies (eg, complement dependent cytotoxicity (CDC) and antibody dependent cellular cytotoxicity (ADCC)). WO 01/90192 describes IgG-like tetravalent antibodies. WO2006 / 020258 describes tetravalent antibodies comprising two diabodies and retaining effector function. Various antigen-binding antibody fragments can be used for the antigen binding site of the bispecific antibody. These include but are not limited to Fv, scFv, and sVD.

Another means of blocking PDGFRβ-mediated signaling is through PDGFRβ-specific small molecule inhibitors. Small molecules represent small organic compounds such as heterocycles, peptides, sugars, steroids and the like. Small molecule modifiers preferably have a molecular weight of less than about 2000 Daltons, preferably less than about 1000 Daltons, more preferably less than about 500 Daltons. Compounds can be modified to enhance efficacy, stability, pharmaceutical suitability, and the like. Small molecule inhibitors include, but are not limited to, small molecules that block ATP binding domains, substrate binding domains, or kinase domains of receptor tyrosine kinases. In another embodiment, the small molecule inhibitor binds to the ligand binding domain of PDGFRβ and blocks receptor activation by the PDGFRβ ligand. Small molecule libraries can be screened for inhibitory activity using high efficiency biochemical, enzymatic, or cell based assays. The assay can be developed to detect the ability of the test compound to inhibit PDGFRβ but not PDGFRα.

Antisense oligodeoxynucleotides, antisense RNAs, and small molecule inhibitory RNAs (siRNAs) provide targeted degradation of mRNA, preventing the translation of proteins. Thus, expression of PDGFRβ can be suppressed. The ability of antisense oligonucleotides to inhibit gene expression was discovered 25 years older than now (Zamecnik and Stephenson, Proc. Natl. Acad. ScI USA. 75: 280-284 (1978)). Antisense oligonucleotides form base pairs with mRNA and pre-mRNA and potentially inhibit several steps of RNA processing and message translation, including splicing, polyadenylation, efflux, stability, and protein translation. (Sazani and Kole, J. Clin. Invest. 112: 481-486 (2003)). However, the two most powerful and widely used antisense methods are the degradation of mRNA or pre-mRNA via RNaseH, and alteration of splicing through targeting aberrant splice junctions. RNaseH recognizes DNA / RNA heterodimers and cleaves RNA at approximately midway between 5 'and 3' of DNA oligonucleotides.

Pioneering RNA-mediated mechanisms can regulate mRNA stability, message translation, and chromatin organization (Mello and Conte, Nature. 431: 338-342, 2004). In addition, long double-stranded RNA (dsRNA) introduced externally is an effective tool for gene silencing in various lower organisms. In mammals, however, long dsRNAs exhibit a highly toxic response that is related to the effects of viral infection and interferon production (Williams, Biochem. Soc. Trans. 25: 509-513, 1997). To avoid this, Elbashir and colleagues (Elbashir et al., Nature 411: 494-498, 2001) report a 5 'phosphate on each strand that selectively degrades the targeted mRNA upon introduction into the cell. And the use of siRNA consisting of a 19-mer duplex with a 3 'overhang of 2 bases.

The action of interfering dsRNA in mammals usually involves two enzymatic steps. First, Dicer (RNase III enzyme) cleaves dsRNA into 21-23-mer siRNA segments. The RNA-induced silent complex (RISC) then unwinds the RNA duplex, pairs one strand with a complementarity region in cognate mRNA, and cleaves at 10 nucleotide sites upstream of the 5 'end of the siRNA strand (Harmon, Nature. 418: 244-251, 2002). Short chemically synthesized siRNAs in the 19-22 mer range do not require a Dicer step and can directly enter the RISC mode. While each strand of the RNA duplex can potentially be loaded onto the RISC complex, it should be understood that the composition of the oligonucleotides can affect the selection of the strands. Thus, for the selective degradation of specific mRNA targets, the duplex should preferentially load the antisense strand component by forming relatively weak base pairs at its 5 'end (Khvorova, Cell. 115: 209-216, 2003). . Exogenous siRNA can be provided as synthesized oligonucleotides or expressed from plasmids or viral vectors (Paddison and Hannon, Curr. Opin. Mol. Ther. 5: 217-224, 2003). In the latter case, the precursor molecule is usually expressed as a short hairpin RNA (shRNA) containing a loop of 4-8 nucleotides and a pillar of 19-30 nucleotides; They are then cleaved by Dicer to form functional siRNAs.

Other means of inhibiting PDGFRβ-mediated signaling include PDGF mimetics that bind but do not activate receptors, and expression of genes or polynucleotides that reduce PDGFRβ levels or activity, such as triple helix inhibitors and dominant negative PDGFRβ mutants. Including but not limited to.

RTK antagonists to be used according to the invention (ie PDGFRβ-specific antagonists, VEGFR antagonists) exhibit one or more of the following properties:

1) Antagonists bind to the external domain of RTK (ie PDGFRβ, VEGFR) and inhibit ligand binding. Inhibition can be determined, for example, by direct binding assays using tablet or membrane bound receptors.

2) Antagonists neutralize receptors. Binding of ligands to external extracellular domains (e.g., PDGF-BB or PDGF-DD to PDGFRβ; VEGF or PlGF to VEGFR) are receptors and downstream signaling molecules such as MAPK, Akt and IRS- Stimulates autophosphorylation of I Neutralization of the receptors includes inhibition, attenuation, inactivation and / or disruption of one or more of these activities normally associated with signaling. Neutralization can be determined, for example, in vivo, in vitro or in vitro using tissue, cultured cells, or purified cell components. Thus, neutralizing PDGFRβ and / or VEGFR inhibits, attenuates, inactivates and / or inhibits growth (proliferation and differentiation), angiogenesis (vascular recruitment, invasion, and metastasis), and cell migration and metastasis (cell adhesion and invasiveness). Or has a variety of effects, including collapse.

One measure of receptor neutralization is the inhibition of tyrosine kinase activity of the receptor. Tyrosine kinase inhibition can be determined using well known methods, for example, by measuring autophosphorylation levels of recombinant kinase receptors, and / or phosphorylation of natural or synthetic substrates. Thus, phosphorylation assays are useful in determining neutralizing antibodies in the context of the present invention. Phosphorylation can be detected, for example, using an antibody specific for phosphotyrosine in an ELISA assay or on a Western blot. Some assays for tyrosine kinase activity are described by Panek et al., J Pharmacol Exp Thera. 283: 1433-44 (1997) and Batley et al., Life Sci. 62: 143-50 (1998). It is described in. Antibodies of the invention reduce the tyrosine phosphorylation of PDGFRβ by at least about 30%, at least about 50%, at least about 75%, preferably at least about 85%, more preferably at least about 90% in cells that respond to the ligand. Cause

Another measure of receptor neutralization is the inhibition of phosphorylation of substrates downstream of receptors or other signaling components. Thus, phosphorylation levels of MAPK, Akt, IRS-I and other cellular components can be measured. The reduction in phosphorylation is at least about 40%, and can be at least about 60%, or at least about 80%.

In addition, methods for detecting protein expression can be used to determine receptor neutralization, where the protein measured is modulated by receptor tyrosine kinase activity. These methods include immunohistochemistry (IHC) for detection of protein expression, fluorescence in situ hybridization (FISH) for detection of gene amplification, competitive radioligand binding assays, solid matrix blotting techniques such as Northern and Southern (Southern) blots, reverse transcriptase polymerase chain reaction (RT-PCR) and ELISA (see, for example, Grandis et al., Cancer, 78: 1284-92 (1996); Shimizu et al. Japan J Cancer Res, 85: 567-71 (1994); Sauter et al., Am J Path., 148: 1047-53 (1996); Collins, Glia 15: 289-96 (1995). Radidinsky et al., Clin Cancer Res. 1: 19-31 (1995); Petrides et al., Cancer Res. 50: 3934-39 (1990); Hoffmann et al., Anticancer Res 17 : 4419-26 (1997); Wikstrand et al., Cancer Res. 55: 3140-48 (1995)). In vitro assays can also be used to determine receptor neutralization. For example, receptor tyrosine kinase inhibition can be observed by mitogenic assays using cell lines stimulated with receptor ligands in the presence and absence of inhibitors. Another method includes testing for inhibition of growth of RTK-expressing tumor cells or cell lines transfected to express PDGFRβ. Inhibition can also be observed using tumor models, eg, human tumor cells injected into mice.

Antagonists of the invention are not limited by any particular receptor neutralizing mechanism. Antagonists of the invention bind externally to cell surface receptors, block the binding of ligands, and subsequent signaling mediated through receptor-associated tyrosine kinases, and prevent phosphorylation of other downstream proteins in PDGFRβ and signaling cascades. Can be.

3) Antagonists downregulate receptors. The amount of RTK present on the surface of a cell is determined by receptor protein production, internalization, and degradation. The amount of receptor present on the surface of a cell can be measured indirectly by detecting the internalization of the receptor or molecules bound to the receptor. For example, receptor internalization can be measured by contacting cells with receptor-specific labeled antibodies. The membrane-bound antibody is then removed, collected and counted. Internalized antibodies are determined by lysing the cells and detecting the label in the lysate.

Another method is to directly measure the amount of receptors present on cells after treatment with anti-RTK antibodies or other substances, for example by fluorescence-activated cell sorting analysis of stained cells for surface expression of RTK. It is. Stained cells are incubated at 37 ° C. and fluorescence intensity is measured over time. As a control, a portion of the stained population can be incubated at 4 ° C. (conditions in which receptor internalization stops).

Cell surface RTKs can be detected and measured using different antibodies that are specific for PDGFRβ and block or do not compete with the binding of the antibody being tested. In certain embodiments, the decrease in cell surface RTK is at least about 50%, or at least about 75%, or at least about 90% in response to treatment with an RTK antagonist. Significant decreases can be observed in as little time as approximately 4 hours.

Another measure of down-regulation is the reduction of total receptor protein present in the cell and reflects the degradation of internal receptors. Thus, treatment of cells (especially cancer cells) with the antibodies of the present invention reduces total cellular RTK. In certain embodiments, the decrease in cell surface RTK is at least about 50%, or at least about 75%, or at least about 90% in response to treatment with an RTK antagonist.

Antibody specificity refers to the selective recognition of antibodies for specific epitopes of antigens. For example, natural antibodies are monospecific. Bispecific antibodies (BsAb) are antibodies with two different antigen-binding specificities or sites. If the antigen-binding protein has more than one specificity, the recognized epitope may be associated with a single antigen or more than one antigen.

Preferably, an antibody of the invention binds to a receptor as strongly as at least a natural ligand binds to that receptor. Affinity (expressed as the equilibrium constant (K _d ) for dissociation of the antigen and the antibody) measures the binding strength between the antigenic determinant and the antibody binding site. Avidity is a measure of the strength of binding between an antibody and its antigen. Avidity relates to both the affinity between the epitope and its antigen binding site on the antibody, and the binding of the antibody. The linker indicates the number of antigen binding sites that the immunoglobulin has for a particular epitope. For example, monovalent antibodies have one binding site for a particular epitope. An antigenic determinant, or epitope, is the site on an antigen to which a given antibody binds. Typical K values are from 10 ⁵ to 10 ⁷ litres / mole. Any K below 10 ⁴ liters / mole is considered to indicate nonspecific binding. The inverse of K is named as K _d . (K _d may also be referred to as a dissociation constant). The smaller the value of K _d, the stronger the binding strength between the antigenic determinant and the antibody binding site.

Antibodies of the invention include any antibody that specifically binds to PDGFRβ and reduces or inhibits receptor mediated signaling. In certain embodiments, the antibody at about 10 ^-8 M ^-1 or less, or about 10 ^-9 M ^-1 or less, or about 3 x 10 ^-10 K _d of less than ^M-1 binding to its respective receptor. Non-limiting examples of certain PDGFRβ-specific antibodies are disclosed and / or illustrated herein. The nucleic acid and amino acid sequences of two specific antibodies of the invention, namely 1B3 and 2C5, are shown in Table 1.

In a preferred embodiment, one, two, three, four, five, or six complementarity determining regions (CDRs) of the antibody have a sequence selected from any one CDR of 1B3. In another preferred embodiment, one, two, three, four, five, or six complementarity determining regions (CDRs) of the antibody have a sequence selected from the group consisting of any one CDR of 2C5.

In another preferred embodiment, an antibody of the invention has a heavy chain variable region sequence that is substantially identical to the heavy chain variable region of 1B3 or 2C5, and / or a light chain variable region sequence that is substantially identical to the light chain variable region of 1B3 or 2C5. "Substantially identical" means that the amino acid sequence is at least 80%, 85%, 90%, 95%, 98%, or 99% identical to the reference sequence.

In an embodiment of the invention, the PDGFRβ-specific antibody comprises a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 18, and a light chain comprising the amino acid sequence of SEQ ID NO: 26. In another embodiment of the invention, the PDGFRβ-specific antibody comprises a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 2, and a light chain comprising the amino acid sequence of SEQ ID NO: 10. PDGFRβ-specific antibodies of the invention further include those that compete with the above-mentioned antibodies (ie, bind to the same or overlapping epitopes).

The present invention also encompasses antibodies having an amino acid sequence substantially identical to the amino acid sequence of the variable or hypervariable region of the aforementioned antibody. Substantially identical amino acid sequences are described herein in Pearson and Lipman, Proc. Natl. Acad. Sci. USA 55: 2444-2448 (1988), as defined by the FASTA search method, defines a sequence having at least about 80%, or at least about 90%, or at least about 95% homology or identity with other amino acid sequences do. It will be expected that various substitutions can be made within the framework region, particularly in light of frequent mutations within the framework region that are observed to be generated from mutations and selection in vivo. However, it is also contemplated that mutations at certain CDR positions will also not be tolerated. For example, not all CDR amino acids are in direct contact with the antigen. Amino acid changes, particularly conservative changes, in the non-contact CDR positions are expected to potentially affect binding affinity better than to abolish all binding. Those skilled in the art can readily make sequence variations by design or randomly and test the results. Conservative amino acid substitutions are defined as changes in amino acid composition by changing one or several amino acids of a peptide, polypeptide or protein or fragment thereof. Substitutions are generally similar properties (eg, acidic, basic, Aromatic, sized, positive or negatively charged, polar, nonpolar). Representative substitutions that can be made for such conservative amino acid substitutions can be made among the following groups of amino acids:

Glycine (G), alanine (A), valine (V), leucine (L) and isoleucine (I);

Aspartic acid (D) and glutamic acid (E);

Alanine (A), serine (S) and threonine (T);

Histidine (H), lysine (K) and arginine (R):

Asparagine (N) and glutamine (Q);

Phenylalanine (F), Tyrosine (Y) and Tryptophan (W)

In accordance with the present invention, PDGFRβ-specific antibodies may be administered alone or in combination with anti-VEGFR antibodies, or may be bispecific antibodies that bind to both PDGFRβ and VEGFR. Examples of VEGFR-specific antibodies are also provided. As discussed above with regard to PDGFRβ-specific antibodies, the disclosed VEGFR antibodies are non-limiting examples.

The invention also provides antibodies that bind to specific domains of PDGFRβ. Antibodies that bind to domains 1 and 2 of PDGFRβ, antibodies that bind to domain 1, and antibodies that bind to domain 2 are included within the scope of the present invention. 2C5 is an example of an antibody that binds to domain 2 of PDGFRβ and domains 1 and 2.

Each variable domain of an antibody of the invention may be a complete immunoglobulin heavy or light chain variable domain, or a functional equivalent or mutant or derivative of a naturally occurring domain, or for example WO 93/11236 (Medical Research Counseling). It may be a synthetic domain constructed in vitro using techniques such as described in Medical Research Council / Griffiths et al. So that the mutation provides a protein or protein fragment that retains antigen-binding activity, including domains corresponding to antibody variable domains including, for example, amino acid substitutions, deletions, additions, amino truncation and carboxy terminal truncation It is possible to let. An important characterization feature is the ability of each variable domain to associate with a complementary variable domain to form an antigen binding site.

Antibodies and fragments of the invention may be linked or fused to additional amino acid residues. Such amino acid residues may possibly be peptide tags that facilitate isolation. Other amino acid residues for the homing of antibodies to specific organs or tissues are also contemplated.

In another aspect of the invention, the antibody or antibody fragment is chemically or biosynthetically linked to an antitumor agent or detectable signal generator, particularly when the antibody is internalized. Antitumor agents linked to an antibody include any substance that destroys or damages the environment in which the antibody is bound or the tumor to which the antibody is bound. For example, antitumor agents are toxic agents, such as chemotherapeutic agents or radioisotopes. Suitable chemotherapeutic agents are known to those skilled in the art and include anthracyclines (eg daunomycin and doxorubicin), methotrexate, bindesin, neocarcinostatin, cis-platinum, chlorambucil, cytosine arabinoside, 5-fluoro Rouridine, melphalan, lysine and calicheamicin. Chemotherapeutic agents are conjugated to the antibody using conventional methods (see, eg, Hermentin and Seiler, Behring Inst. Mitt. 52: 197-215 (1988)).

Detectable signal generators are useful in vivo and in vitro for diagnostic purposes. The signal generator produces a measurable signal detectable by external means, usually by measurement of electromagnetic radiation. In general, the signal generating agent is an enzyme or chromophore or emits light by fluorescence, phosphorescence or chemiluminescence. Chromophores include dyes that absorb light in the ultraviolet or visible region and may be substrates or degradation products of enzyme catalyzed reactions.

The present invention further contemplates antibodies to which a target or reporter moiety is linked. The target moiety is the first member of the binding pair. The antitumor agent is conjugated to, for example, a second member of such a pair, leading to the site where the antigen-binding protein is bound. Typical examples of such binding pairs are avidin and biotin. In a preferred embodiment, biotin is conjugated to the antigen-binding protein of the invention to provide a target for an anti-tumor agent or other moiety conjugated to avidin or streptavidin. Alternatively, biotin or other such moieties are linked to the antigen-binding proteins of the invention and used as reporters in diagnostic systems, for example, where a detectable signal generator is conjugated to avidin or streptavidin.

Suitable radioisotopes for use as antitumor agents are also known to those skilled in the art. For example, 131I or 211At is used. These isotopes are attached to antibodies using conventional techniques (see, eg, Pedley et al, Br. J. Cancer 68: 69-73 (1993)). Alternatively, the antitumor agent attached to the antibody is an enzyme that activates the prodrug. In this manner, the prodrug is administered, which remains inactive until reaching the tumor site, converted to its cytotoxin at the tumor site, and the antibody complex is administered. In practice, antibody-enzyme conjugates are administered to the patient and localized within the region of the tissue to be treated. The prodrug is then administered to the patient such that conversion to cytotoxic drugs occurs in the region of the tissue to be treated. Alternatively, the antitumor agent conjugated to the antibody is a cytokine such as interleukin-2 (IL-2), interleukin-4 (IL-4) or tumor necrosis factor alpha (TNF-α). Antibodies target cytokines to tumors, allowing them to mediate damage or destruction of tumors without affecting other tissues. Cytokines are fused to antibodies at the DNA level using conventional recombinant DNA techniques.

The invention also provides an isolated polynucleotide encoding the antibody or fragment thereof described above. The present invention includes nucleic acids having sequences encoding PDGFRβ-specific antibodies and antigen binding fragments comprising all CDRs of 1, 2, 3, 4, 5 and / or 6 described in Table 1.

Accordingly, the present invention provides nucleic acids that specifically hybridize (or specifically bind) to SEQ ID NO: 1, SEQ ID NO: 9, SEQ ID NO: 17, and SEQ ID NO: 25 under stringent hybridization conditions. Without the degeneracy of the nucleic acid code, nucleic acids that specifically bind to the sequences mentioned above are also contemplated. The nucleic acid may be of sufficient length to encode a complete protein (eg, complete V _H or V _L ) or fragment thereof.

Hybridization under stringent conditions refers to conditions under which the probe preferentially hybridizes to its target subsequence and less to other sequences or to none at all. It will also be understood that stringent hybridization and stringent hybridization wash conditions in the context of nucleic acid hybridization experiments, eg, Southern and Northern hybridization, are sequence dependent and different under different environmental parameters. It is known in the art to adjust the hybridization and wash solution content and temperature to achieve stringent hybridization conditions. Stringency is determined by parameters such as the size of the probe used and the nucleotide content. For general descriptions and examples, see Sambrook et al., 1989, Molecular Cloning-A Laboratory Manual (2nd ed.) Vol. 1-3, Cold Spring Harbor Laboratory, Cold Spring Harbor Press, NY] and other sources. Other guidelines for hybridization of nucleic acids can be found in Tijssen, 1993, Laboratory Techniques in Biochemistry and Molecular Biology--Hybridization with Nucleic Acid Probes, part I, chapter 2, Overview of principles of hybridization and the strategy of nucleic acid probe assays, Elsevier , NY].

Preferred stringent conditions are that the probe hybridizes to sequences that are more than about 90% complementary to the probe and not to sequences that are complementary to less than about 70%. In general, highly stringent hybridization and wash conditions are selected to be about 5 ° C. lower than the heat melting point (T _m ) for the specific sequence at the defined ionic strength and pH. T _m is the temperature at which 50% of the target sequence hybridizes to a correctly matched probe (at defined ionic strength and pH). Very stringent conditions are chosen to be equal to T _m for the particular probe.

An example of stringent hybridization conditions for hybridization of complementary nucleic acids with more than 100 complementary residues on a filter in a Southern or Northern blot is 50% formamide in the presence of 1 mg of heparin at 42 ° C., where hybridization is performed overnight. An example of highly stringent washing conditions is 0.15 M NaCl for about 15 minutes at 72 ° C. An example of stringent washing conditions is a 0.2 × SSC wash for 15 minutes at 65 ° C. (Sambrook et al., 1989). Often, a low stringency wash is performed before a high stringency wash to remove background probe signal. For example, an example of an intermediate stringency wash for duplexes of more than 100 nucleotides is 1 x SSC for 15 minutes at 45 ° C. For example, an example of a low stringency wash for duplexes of more than 100 nucleotides is 4-6 x SSC for 15 minutes at 40 ° C. In general, signal to noise ratios that are twice (or more) than those observed for unrelated probes in certain hybridization assays indicate detection of specific hybridization.

Nucleic acids that do not hybridize to each other under stringent conditions are still substantially identical if the polypeptides they encode are substantially identical. This occurs, for example, when the nucleic acid is produced using the maximum codon multiplicity allowed by the genetic code. Thus, the nucleotide sequence of the present invention comprises a sequence of nucleotides that are at least about 70%, preferably at least about 80%, more preferably at least about 90% identical to SEQ ID NO: 1, SEQ ID NO: 9, SEQ ID NO: 17, or SEQ ID NO: 25. The invention also provides a recombinant vector containing a nucleic acid of the invention. The vector can be an expression vector, wherein the nucleic acid is operably linked to a control sequence, eg, a promoter and optionally an enhancer sequence. Various expression vectors have been developed for efficient synthesis of antibody polypeptides in prokaryotic and eukaryotic systems, including but not limited to yeast and mammalian cell culture systems.

Any suitable expression vector can be used. Expression vectors useful in the present invention contain at least one expression control sequence operably linked to the DNA sequence or fragment to be expressed. Control sequences are inserted into the vector to control and regulate the expression of the cloned DNA sequence. Examples of useful expression control sequences include the lac system, trp system, tac system, trc system, major operator and promoter regions of phage lambda, control regions of fd coat protein, glycosylation promoters of yeast, eg, 3-phosphoglycer Promoters for late kinase, promoters of yeast acid phosphatase, such as Pho5, promoters of yeast alpha-crossing factors, and promoters derived from polyomas, adenoviruses, retroviruses, and monkey viruses, such as SV40 Early and late promoters, and other sequences known to control the expression of genes of prokaryotic or eukaryotic cells and their viruses or combinations thereof.

Selectable markers are genes that encode proteins essential for the survival or growth of transformed host cells growing in selective culture medium. Typical selectable markers are (a) confer resistance to antibiotics or other toxins such as ampicillin, neomycin, methotrexate or tetracycline, (b) supplement autotrophic deficiency, or (c) be available from complex media There is a gene that encodes a protein that supplements important nutrients that are not, and that encodes, for example, D-alanine racemase for Bacillus . Particularly useful selectable markers confer resistance to methotrexate. For example, cells transformed with the DHFR selection gene are first identified by culturing all transformants in a culture medium containing methotrexate (Mtx), a competitive antagonist of DHFR. Suitable host cells when wild type DHFR are used are described in Urlaub and Chasin (1980) Proc. Natl. Acad. Sci. USA 77, 4216, which is a Chinese hamster ovary (CHO) cell line lacking DHFR activity that has been produced and expanded. The transformed cells are then exposed to increased levels of methotrexate. This results in the synthesis of multiple copies of the DHFR gene and DNA encoding multiple copies of other DNA, antibodies, or antibody fragments, including expression vectors.

If expression of gene constructs in yeast is desired, examples of suitable selection genes for use in yeast are the trp1 gene present in yeast plasmid YRp7 (Stinchcomb et al. (1979) Nature, 282, 39; Kingsman et al. (1979) Gene 7, 141). trp1 gene is a mutant strain of yeast that lacks the ability to grow on tryptophan, eg, ATCC No. Provide a selection marker for 44076 or PEP4-1 (Jones (1977) Genetics 85, 12). The presence of trp1 damage in the yeast host cell genome then provides an effective environment for detecting transformation by growing in the absence of tryptophan. Similarly, Leu2-deficient yeast strains (ATCC 20,622 or 38,626) are supplemented by known plasmids carrying the Leu2 gene. An example of a vector useful in yeast is a 2μ plasmid.

Examples of prokaryotic cloning vectors are as follows. Plasmids from E. coli, for example colE1, pCR1, pBR322, pMB9, pUC, pKSM and RP4. Prokaryotic vectors also include derivatives of phage DNA, such as M13 and other filamentary single stranded DNA phages. Certain antibodies can be expressed using DNA constructs comprising bacterial secretion signal sequences at the beginning of each polypeptide chain. Produced conveniently in E. coli. Various bacterial signal sequences are known in the art. Preferred signal sequences are Erwinia carotovora ) from the pelB gene.

Suitable vectors for expression in mammalian cells include shuttle vectors derived from combinations of well known derivatives of SV40, adenoviruses, retrovirus-derived DNA sequences, and functional mammalian vectors, such as those described above, And functional plasmids and phage DNA. DNA fragments encoding the antibodies can be cloned into vectors using, for example, human cytomegalovirus (HCMV) promoters and enhancers for high level expression in mammalian cells (see, eg, US Pat. No. 5,840,299 to Bendig, et. Maeda, et al (1991) Hum. Antibod. Hybridomas 2, 124-34; PJ Southern and P. Berg, J. Mol. Appl. Genet. 1: 327-41 (1982); Subramani et al., Mol. Cell. Biol. 1: 854-64 (1981); Kaufmann and Sharp, "Amplification And Expression of Sequences Cotransfected with a Modular Dihydro folate Reductase Complementary DNA Gene," J. Mol. Biol 159: 601-21 (1982); Kaufmann and Sharp, Mol. Cell. Biol. 159: 601-64 (1982); Scahill et al., "Expression And Characterization Of The Product Of A Human Immune Interferon DNA Gene In Chinese Hamster Ovary Cells, "Proc. Nat'l Acad. Sci. USA 80, 4654-59 (1983); Urlaub and Chasin, Proc. Nat'l Acad. Sci. USA 77: 4216-20 ( 1980).

The present invention also provides a recombinant host cell containing the recombinant or expression vector described above. Particularly preferred cell lines are selected based on high levels of expression, constitutive expression of the protein of interest and minimal contamination from the host protein. Useful prokaryotic hosts are for example E. coli. Coli, for example Lee. E. coli SG-936, Lee. E. coli HB 101, Lee. E. coli W3110, Yi. E. coli X1776, this. E. coli X2282, there. E. coli DHI and Yi. E. coli MRC1, Pseudomonas (Pseudomonas), Bacillus, such as Bacillus subtilis (Bacillus It includes subtilis), and Streptomyces (Streptomyces). Mammalian cell lines available as hosts for expression are well known in the art and include many immortalized cell lines, including but not limited to COS-7 cells, Chinese hamster ovary (CHO) cells, baby hamster kidney (BHK) cells, PER.C6 cells, and many others, including cell lines of lymphatic origin, such as lymphoma, myeloma, or hybridoma cells. Suitable further eukaryotic cells include yeast and other fungi.

Transformed host cells are assimilable carbon sources such as carbohydrates such as glucose or lactose, nitrogen such as amino acids, peptides, proteins or their degradation products such as peptone, ammonium salts and the like and inorganic salts such as In a liquid medium containing, for example, sulfates, phosphates and / or carbonates of sodium, potassium, magnesium and calcium. The medium further contains, for example, growth promoting substances, for example trace elements such as iron, zinc, manganese and the like.

The invention also provides a method of producing an antibody comprising culturing the host cell under conditions that allow expression of the antibody. After expression in host cells maintained in a suitable medium, the antibody can be isolated and purified from the medium by methods known in the art.

The invention further provides a pharmaceutical composition comprising an antibody, nucleic acid, vector or host cell of the invention and a pharmaceutically acceptable carrier.

The invention also includes treating PD or inhibiting tumor growth, treating or inhibiting angiogenesis-dependent conditions (eg, tumor growth) in a mammal, comprising administering PDGFRβ-specific antagonists, It relates to a method for treating a neoplastic disease. In one embodiment, the antagonist blocks PDGFRβ-mediated stimulation of tumor cells. In another embodiment, the antagonist functions to inhibit or prevent angiogenesis, thereby treating or inhibiting angiogenesis-dependent conditions. In general, the vascular endothelium is stimulated in a lateral secretory manner by growth factors from other sources (eg, tumor cells). Thus, PDGFRβ antagonists are effective to treat subjects with vascularized tumors or neoplasms.

"Treating" a disease includes (1) preventing the disease from developing in a mammal that may be predisposed to the disease but has not yet experienced or exhibited symptoms of the disease; For example, to prevent the development of clinical symptoms; (2) inhibit, for example, stop or retard its development; (3) to alleviate the disease, including causing regression of symptoms of the disease.

Tumors that can be treated include refractory tumors as well as primary and metastatic tumors. Refractory tumors include tumors that do not respond or are resistant to treatment with chemotherapeutic agents alone, antibodies alone, radiation alone, or a combination thereof. Refractory tumors also include tumors that appear to be inhibited by treatment with such agents but relapse up to 5 years, sometimes up to 10 years after discontinuation of treatment.

PDGFRβ-specific antagonists are administered at a sufficient dose and frequency of administration to substantially saturate the target receptor or ligand thereof. Substantial saturation is at least about 50%, or at least about 80%, or at least about 95% saturation of the targeted receptor. PDGFRβ-specific antagonists are administered at a frequency sufficient to maintain substantial saturation for at least about 50%, or at least about 70%, or at least about 90% of the intervals between doses. In an embodiment of the invention, the PDGFRβ-specific antagonist is an antibody. For treatment, the dosage is about 5 mg / m ² to about 700 mg / m ² . In another embodiment, the dosage is about 10 mg / m ² to about 250 mg / m ² . Appropriate dosages and schedules can be readily determined by one skilled in the art using, for example, the concentrations required to achieve receptor saturation or neutralization in vitro, or by analysis of serum concentrations of antagonists.

In an embodiment of the invention, the PDGFRβ-specific antagonist is administered in combination with a chemotherapeutic agent. Chemotherapeutic agents known or evaluated in the art may be classified based on their target or mode of action. For example, alkylating agents include but are not limited to cisplatin, cyclophosphamide, melphalan and dacarbazine. Examples of anti-metabolites are doxorubicin, daunorubicin and paclitaxel, gemcitabine, and topoisomerase inhibitor irinotecan (CPT-11), aminocamptothecin, camptothecin, DX-8951f, and topotecan (topoimerase) I) and etoposide (VP-16) and teniposide (VM-26) (topoisomerase II). Other suitable chemotherapeutic agents are known to those of skill in the art and include methotrexate, binddesin, neocarzinoststatin, chlorambucil, cytosine arabinosides, 5-fluorouridine, melphalan, lysine and calicheamicin. PDGFRβ antagonists and chemotherapeutic agents are administered to the patient in an amount effective to inhibit angiogenesis and / or reduce tumor growth. PDGFRβ antagonists may also be administered in combination with other treatment regimens, such as radiation therapy. For radiation, the source can be external (external beam radiation therapy-EBRT) or internal (proximal therapy-BT) for the patient being treated.

The dosage of the anti-neoplastic agent to be administered depends on a number of factors, including, for example, the type of substance, the type and depth of the tumor being treated, and the route of administration of the substance. However, it should be emphasized that the present invention is not limited to any particular dosage.

In an embodiment of the invention, the PDGFRβ-specific antagonist is administered in combination with a VEGFR antagonist. Examples of VEGFR antagonists include antibodies that bind VEGFR2 / KDR, such as IMC-2C6 (nucleotides and amino acid sequences of V _H : SEQ ID NOs: 33 and 34; nucleotides and amino acid sequences of V _L : SEQ ID NOs: 35 and 36) (WO 03 / 075840) and IMC-1121 (nucleotide and amino acid sequences of V _H : SEQ ID NOs: 33 and 34; nucleotide and amino acid sequences of V _L : SEQ ID NOs: 37 and 38) (see WO 03/075840). Examples of antibodies that bind VEGFR1 / Flt-1 include 6.12 (nucleotide and amino acid sequences of V _H : SEQ ID NOs: 39 and 40; nucleotide and amino acid sequences of V _L : SEQ ID NOs: 41 and 42) and IMC-18F1 (nucleotides of V _H and Amino acid sequences: SEQ ID NOs: 43 and 44; nucleotides of V _L and amino acid sequences: SEQ ID NOs: 45 and 46). An example of an antibody specific for VEGF is Avastin®.

As disclosed above for PDGFRβ-specific antibodies, VEGFR antagonists reduce or inhibit signaling mediated by the VEGF receptor. Inhibitory mechanisms include, but are not limited to, ligand blocking, receptor dimer or multimer formation, receptor internalization, inhibition of enzymatic activity (eg, autophosphorylation, modification of receptor substrate), and the like.

In certain embodiments, the antibodies of the invention have bispecificity and can bind to two different antigens simultaneously. Different antigens can be located on different cells or on the same cell. Crosslinking of an antigen, for example, provides a solid support to which the first antigen is bound, adds a bispecific antibody specific for the first antigen, and the binding protein also adds a specific second antigen, It can be confirmed in vitro by detecting the presence of a second antigen.

Preferred antibodies of the invention can block the interaction between two receptors and their respective ligands. For example, antibodies specific for PDGFRβ and KDR inhibit PDGF-BB induced cell activation and VEGF or PlGF induced cell migration. Compared to monospecific antibodies, bispecific antibodies can be more potent inhibitors of cellular function.

In one aspect of the invention, the PDGFRβ antagonist is co-administered with the VEGFR antagonist. The method is effective for treating solid or non-solid tumors, for example tumors that are not vascularized or are not yet substantially vascularized.

Tumors and neoplasms that can be treated using PDGFRβ antagonists alone or in combination with VEGFR antagonists include, for example, malignant tumors and neoplasms, such as blastomas, carcinomas or sarcomas, and highly vascular tumors and neoplasms. do. Cancers that can be treated by the methods of the invention include cancers of the brain, urogenital tract, lymphatic system, stomach, kidneys, colon, larynx and lung and bone. Non-limiting examples include epidermal tumors, squamous cell tumors such as head and neck tumors, colorectal tumors, prostate tumors, breast tumors, lung tumors such as lung adenocarcinomas and small cell and non-small cell lung tumors, pancreatic tumors, thyroid gland Tumors, ovarian tumors and liver tumors. The composition can be used for the treatment of vascularized skin cancers such as squamous cell carcinoma, basal cell carcinoma and skin cancers that can be treated by growth inhibition of malignant keratinocytes, eg human malignant keratinocytes. Other cancers that can be treated include Kaposi's sarcoma, CNS neoplasia (neuroblastoma, capillary blastoma, meningioma and brain metastases), melanoma, gastrointestinal and kidney carcinoma and sarcoma, rhabdomyosarcoma, glioblastoma, for example Glioblastoma multiforme, and leiomyosarcoma.

Examples of non-solid tumors include leukemia, multiple myeloma and lymphoma. Some examples of leukemia include acute myeloid leukemia (AML), chronic myeloid leukemia (CML), acute lymphocytic leukemia (ALL), chronic lymphocytic leukemia (CLL), erythroblastic leukemia or mononuclear leukemia. Some examples of lymphomas include Hodgkin and non-Hodgkin's lymphomas.

The compositions of the present invention also include conditions that are characterized, for example, by hyperangiogenesis involving vascularization and / or inflammation, such as atherosclerosis, rheumatoid arthritis (RA), neovascular glaucoma, proliferative retinopathy, eg It may be used for the treatment or prevention of proliferative diabetic retinopathy, macular degeneration, hemangioma, hemangiofibroma and psoriasis. Other non-limiting examples of non-neoplastic angiogenic diseases include retinopathy of premature infants (Posterior lens fibrosis), corneal graft rejection, insulin-dependent diabetes, multiple sclerosis, myasthenia gravis, Chron's disease, autoimmune nephritis, Primary biliary cure, acute pancreatitis, allograft rejection, allergic inflammation, contact dermatitis and delayed hypersensitivity reactions, inflammatory bowel disease, septic shock, osteoporosis, osteoarthritis, cognitive decline caused by neuronal inflammation, Osler-Weber (Osler-Weber Weber) syndrome, restenosis, and fungal, parasitic and viral infections such as cytomegalovirus infection.

Identification of such diseases can be performed with the skills and knowledge of those skilled in the art. For example, human subjects suffering from clinically significant neoplastic or angiogenic diseases or at risk of developing clinically significant symptoms are suitable for administration of PDGFRβ antagonists, optionally in combination with VEGFR antagonists. The skilled clinician can easily determine whether an individual is a candidate for such treatment, for example using clinical trials, physical examinations and medical / family history.

The antagonist composition of the present invention may be administered in an amount sufficient to prevent, inhibit or reduce the progression of the tumor or condition for therapeutic treatment for a patient suffering from a condition involving tumor or angiogenesis. Progression includes, for example, growth, invasiveness, metastasis and / or recurrence of a tumor or condition. Appropriate amounts to achieve these are defined as therapeutically effective dosages. The amount effective for this use will depend on the severity of the disease and the overall condition of the patient's own immune system. The schedule of administration will also vary depending on the condition of the disease and the condition of the patient, and generally ranges from a single bolus dose or continuous infusion to multiple doses daily (eg, every 4-6 hours), or of a treating physician. And the range indicated by the patient's condition. However, it should be understood that the present invention is not limited to any particular dosage.

Methods of administration to mammals include, for example, oral, intravenous, intraperitoneal, subcutaneous or intramuscular administration.

PDGFRβ-specific and VEGFR-specific antibodies can be conjugated chemically or biosynthetically to other substances, eg anti-neoplastic or anti-angiogenic agents, for the treatment of diseases. Antitumor agents linked to an antibody include any substance that destroys or damages the environment in which the antibody is bound or the tumor to which the antibody is bound. For example, antitumor agents are toxic agents, such as chemotherapeutic agents or radioisotopes. Chemotherapeutic agents are conjugated to antibodies using conventional methods, including peptides and non-peptide linkers (see, eg, Hermentin and Seiler (1988) Behring Inst. Mitt. 82, 197-215).

PDGFRβ-specific antibodies of the invention may also be linked to detectable signal generators useful in vivo and in vitro for diagnostic purposes. The signal generator produces a measurable signal detectable by external means, usually by measurement of electromagnetic radiation. In general, the signal generating agent is an enzyme or chromophore or emits light by fluorescence, phosphorescence or chemiluminescence. Chromophores include dyes that absorb light in the ultraviolet or visible region and may be substrates or degradation products of enzyme catalyzed reactions.

The present invention further contemplates the use of the antibody with a therapeutic or diagnostic agent incorporated into the second reagent. For example, one member of a binding pair is linked to an antibody of the invention. Anti-neoplastic agents are conjugated to, for example, the second member of the pair, leading to the site to which the antibody binds. In a preferred embodiment, biotin is conjugated to an antibody of the invention to provide a target for an anti-neoplastic agent or other moiety conjugated to avidin or streptavidin. Alternatively, biotin or other such moieties are linked to the antibodies of the invention and used, for example, as reporters in diagnostic systems in which a detectable signal generator is conjugated to avidin or streptavidin.

PDGFRβ antagonists of the invention, optionally in combination with VEGFR antagonists, include, but are not limited to, one or more suitable adjuvants such as cytokines (eg IL-10 and IL-13) or other immune stimulators, such as, but not limited to It can be administered in combination with chemokines, tumor associated antigens and peptides. However, it should be appreciated that administration of the antibody alone is sufficient to prevent, inhibit or reduce the progression of the tumor in a therapeutically effective manner.

In certain embodiments, it may be desirable to administer the antibodies of the invention that bind RTK and block ligand binding in combination with other antigen-binding proteins that bind ligand. Ligand binding antibodies are known in the art and include, for example, anti-VEGF (Avastin®; Bevacizumab).

In accordance with the present invention, the co-administration of PDGFRβ-specific antagonists and VEGFR antagonists may be accompanied by the administration of anti-neoplastic agents such as chemotherapeutic agents or radioisotopes. Examples of useful anti-neoplastic agents include those described above for co-administration with PDGFRβ-specific antagonists.

In combination therapy, the PDGFRβ antagonist and VEGFR antagonist may be co-administered separately or together on the same day on different schedules. Optionally, the antagonist may be combined into a single dose. Optionally, the antagonist may be combined into a single active entity (eg, bispecific antibody). PDGFRβ antagonist / VEGFR antagonist therapy may be used before, during or after the therapy with other agents, as well as in any combination thereof, ie, before and during therapy with anti-neoplastic agents, Before and after start, during use and after start, or before start, during use and after start. For example, for the treatment of tumors or neoplastic diseases, PDGFRβ antagonist / VEGFR antagonist therapy may be used 1-30 days before the radiation therapy, preferably 3-20 days before, more preferably 5-12 days before. May be administered. In accordance with the present invention, chemotherapy or radiation therapy is administered concurrently with, prior to, or following PDGFRβ antagonist / VEGFR antagonist therapy.

In the present invention, any suitable method or route may be used to administer the antibodies of the present invention and optionally to co-administer anti-neoplastic agents, receptor antagonists, or other pharmaceutical compositions. For example, anti-neoplastic therapies used in accordance with the present invention include any therapy that is considered optimally suitable for the treatment of neoplastic conditions in a patient. Different malignants may require the use of specific anti-tumor antibodies and specific anti-neoplastic agents, which will be determined based on the individual patient. Routes of administration include, for example, oral, intravenous, intraperitoneal, subcutaneous, or intramuscular administration. The dosage of the anti-neoplastic agent to be administered depends on a number of factors including, for example, the type of anti-neoplastic agent, the type and depth of tumor to be treated and the route of administration of the anti-neoplastic agent. However, it should be emphasized that the present invention is not limited to any particular method or route of administration.

It is contemplated that the antibodies of the present invention will be administered in the form of a composition further comprising a pharmaceutically acceptable carrier when used in a mammal for the purpose of prophylaxis or treatment. Suitable pharmaceutically acceptable carriers include, for example, one or more of water, saline, phosphate buffered saline, dextrose, glycerol, ethanol and the like and combinations thereof. Pharmaceutically acceptable carriers may further comprise small amounts of adjuvants, such as wetting or emulsifying agents, preservatives or buffers, which improve the shelf life or effectiveness of the binding protein. Injectable compositions can be formulated to provide rapid, sustained or delayed release of the active ingredient after administration to a mammal, as is known in the art.

The invention also includes a kit for inhibiting tumor growth and / or angiogenesis, comprising a therapeutically effective amount of a PDGFRβ-specific antagonist of the invention. In an embodiment of the invention, the kit further comprises a VEGFR antagonist (eg, an antagonist of VEGF, PlGF, VEGFR-1 / Flt-1, VEGFR-2 / Flk-1 / KDR, or VEGFR3 / Flt-4). Include. VEGF antagonists can be, for example, small molecules, ligand-binding receptor fragments, or antibodies. Where the antagonist administered to human is an antibody, human, humanized, or chimeric antibodies are preferred. The kit may further contain any suitable antagonist of other growth factor receptors (eg, EGFR, IGFR, NGFR, FGFR, etc. as described above) involved in tumorigenesis or angiogenesis. Alternatively or in addition, the kits of the present invention may further comprise an anti-neoplastic agent. Examples of anti-neoplastic agents suitable in the context of the present invention are provided herein. Kits of the invention may further comprise an adjuvant; Examples are also described above.

Use of the antibodies of the invention in vivo and in vitro for investigational or diagnostic methods well known in the art is also included within the scope of the invention. Diagnostic methods include kits containing the antibodies of the invention.

Thus, the receptor binding antibodies of the invention can be used in vivo and in vitro for methods of investigation, diagnosis, prophylaxis, or treatment well known in the art. Of course, it should be understood and expected that variations in the principles of the invention disclosed herein may be made by those skilled in the art, and such modifications are intended to be included within the scope of the invention.

All references mentioned herein are incorporated by reference in their entirety.

The following examples further illustrate the invention, but should not be construed to limit the scope of the invention in any way. Detailed descriptions of conventional methods such as used in the construction of vectors and plasmids, insertion of genes encoding polypeptides into said vectors and plasmids, introduction of plasmids into host cells, and expression of genes and gene products and their determination are given in the literature. (Sambrook, J. et al., (1989) Molecular Cloning: A Laboratory Manual, 2 ^nd ed., Cold Spring Harbor Laboratory Press); and Colingan, J. et al. (1994) Current Protocols in Immunology, Wiley & Sons, Incorporated].

Phage Display Library . Human Fab phage display libraries containing 3.7 × 10 ¹⁰ clones (de Haard, HJ et al., 1999, J. Biol. Chem. 274: 18218-30) were used in the literature using immobilized mPDGFRβ-Fc. According to the described procedure (Lu, D. et al., 2002, Int. J. Cancer 97: 393-9; Zhu, Z. et al., 1998, Cancer Res. 58: 3209-14); -mPDGFRβ antibodies were screened. Individual phage clones recovered after selection of the second and third rounds were examined for binding to immobilized mPDGFRβ-Fc by ELISA. To generate soluble Fabs, plasmids of individual binders were used to identify non-inhibiting cells. E. coli host HB2151 was transformed. Soluble Fab proteins were purified from periplasmic extracts by affinity chromatography using a Protein G column (Amersham Pharmacia Biotech, Piscataway, NJ) according to the manufacturer's protocol.

Quantitative Receptor Binding and Blocking Assay . In binding assays, varying amounts of 1B3 IgG or Fab were added to mPDGFRβ-coated plates (50 μl at 1 μg / ml) and incubated for 1 h at RT before the plates were washed three times with PBST. Plates were then incubated with goat anti-human-κ antibody-horse radish peroxidase (HRP) conjugate (Jackson ImmunoResearch, West Grove, Pa.) For an additional 1 h at RT. . Plates were washed and developed as described in the literature (Lu, D. et al., 2002; Zhu, Z. et al., 1998). In blocking assays, various amounts of purified antibody were first mixed with a fixed amount of mPDGFRβ (50 ng at 0.5 μg / ml) and incubated for 30 minutes at RT. The mixture was then transferred to 96-well plates precoated with PDGF-BB (0.5 μg / ml) and incubated for 1 h at RT. After three washes with PBST, the plates were incubated with goat anti-human Fc antibody-HRP conjugate (Jackson Immunoresearch) for 1 h and developed as described in the literature (Lu, D. et al. , 2002]; [Zhu, Z. et al., 1998]; [Loizos, N. et al., 2005]. IC ₅₀ (an antibody concentration that blocks 50% of mPDGFRβ binding to its ligand) was determined.

Antibody affinity determination. Binding kinetics of various Fab and IgG to mPDGFRβ were measured using the BIAcore 3000 biosensor (BIACORE, Inc., Uppsala, Sweden). Briefly, mPDGFRβ was immobilized on a sensor chip and soluble antibodies were injected at a concentration of 1.5 nM to 100 nM. Sensorgrams were obtained at each concentration and evaluated using the program BIA Evaluation 2.0. The affinity constant K _d was calculated from the ratio of dissociation rate (koff) / association rate (kon) ([Lu, D. et al., 2002]; [Zhu, Z. et al., 1998]; [Loizos , N. et al., 2005).

Selection of Human Anti- mPDGFR β Antibodies . 190 clones from the second round and 95 clones from the third round were randomly selected and tested for mPDGFRβ binding and blocking activity by both phage ELISA and soluble Fab ELISA. More than 72% clones from the second round and more than 98% clones from the third round were found to bind specifically to mPDGFRβ, suggesting a high efficiency of the selection process. Ligand blocking analysis showed that about 2.5% of the binder also blocked mPDGFRβ binding to its ligand PDGF-BB.

All seven Fab fragments specifically bind mPDGFRβ and blocked mPDGFRβ with its ligand PDGF-BB with different efficacy. IC ₅₀ values (ie, antibody concentrations required to block 50% of PDGFRβ / PDGF-BB interactions) were between about 4 nM and> 33 nM (FIG. 1B). Clone 1B3 (Table 1), a clone with the best receptor binding efficiency and receptor / ligand blocking efficacy was selected for further study.

Full length IgG antibody cloning and expression . DNA sequences encoding the heavy and light chain genes of the leading clone 1B3 were amplified by PCR and cloned into expression vectors containing human κ light chain constant region and human γ1 heavy chain constant region. Expression vectors were transfected into NS0 myeloma cells and stable clones of 1B3-expressing cells were selected. Cells were grown in serum free medium and full length 1B3 IgG was purified from cell culture supernatants by protein A affinity chromatography (Poros A, Applied Biosystems, Foster City, CA, USA).

As expected, 1B3 IgG showed much better binding efficiency to immobilized mPDGFRβ than its Fab fragment. IC ₅₀ values were 0.34 nM for 1B3 IgG and 1.3 nM for 1B3 Fab (FIG. 1A). Binding kinetics analysis by surface plasmon resonance using the BIAcore instrument showed that the affinity of 1B3 for binding to mPDGFRβ was about 57-fold higher than its Fab form (5.1 nM for Fab and 0.09 nM for IgG). No cross-reactivity was observed when the 1B3 antibody was tested for binding to human PDGFRβ as a soluble recombinant protein on human tumor cell lines or as cell-surface expressed receptor. In addition, 1B3 did not bind to human and mouse PDGFRα (data not shown). In the blocking assay, 1B3 inhibited the binding of mPDGFRβ to its ligand PDGF-BB with an IC ₅₀ of 1.2 nM, whereas the IC ₅₀ of its Fab was 4.1 nM (FIG. 1B).

1B3 inhibits PDGF - BB -stimulated receptor phosphorylation and downstream signaling in cell based assays . Several mouse cell lines such as NIH / 3T3 (fibroblasts), D122 (Lewis lung carcinoma), 4T1 (breast cancer), B16 (melanoma) and H5V (PmT-transformed endothelial cells) were used as test agents Was tested for PDGFRβ expression by FACS analysis. Cells are aliquoted at about 1 × 10 ⁶ / well into wells of a 96-well plate and incubated with 1B3 (10 μg / ml) for 1 h at 4 ° C., followed by anti-human Fc antibody-FITC conjugate (Jackson Immunoresearch Incubated at 4 ° C. for an additional hour. After washing several times with cold PBS, cells were analyzed using a FACSyantage SE flow cytometer (BD Biosciences, San Jose, Calif.). Anti-mPDGFRβ antibody of R & D was used as a positive control (data not shown). FACS results (FIG. 2A) show that NIH / 3T3 and D122 express PDGFRβ at moderate levels, 4T1 and B16 express receptors at low levels, and H5V did not show any staining.

Receptor phosphorylation was determined for NIH / 3T3 and D122 cells. Cells were plated on 6 cm dishes and grown to 70-80% confluence, then cells were washed twice in PBS and incubated overnight in serum free medium. Cells were first incubated with various antibodies at RT for 30 minutes and then stimulated with PDGF-BB at 37 ° C. for 15 minutes. Cells were dissolved in lysis buffer (50 mM Tris-HCl, pH 7.4), 150 mM NaCl, 1% TritonX-100, 1 mM EDTA, 1 mM phenylmethylsulfonyl fluoride, 0.5 mM Na ₃ VO ₄ , 1 μg / ml After dissolving for 1 h in peptin, 1 μg / ml pepstatin, and 1 μg / ml aprotinin), the lysates were centrifuged at 4 ° C. for 10 min at 12,000 rpm. The receptor was immunoprecipitated from the cell lysate supernatant with anti-mPDGFRβ antibody (R & D Systems Inc), followed by the addition of 20 μl of ProA / G-Sepharose beads. Precipitated receptor protein was dissolved on a 4-12% NuPAGE Bis-Tris gel and transferred to a polyvinylidene difluoride membrane. Phospho-mPDGFRβ protein was detected on blots using anti-phospho-tyrosine antibody-HRP conjugate (Santa Cruz Biotech, Santa Cruz, Calif.). Total receptor protein loaded on the gel was analyzed using an antibody against mPDGFRβ.

To study downstream signaling, cell lysates from the treatment were lysed by 4-12% NuPAGE Bis-Tris gel. Phosphorylated Akt and p44 / 42 mitogen-activated protein kinase (MAPK) were treated with anti-phospho-Akt and anti-phospho-p44 / p42 MAPK and p44 / p42 MAPK (eBioscience, California, USA). San Diego)).

1B3 inhibited PDGF-BB-stimulated receptor phosphorylation in both dose dependent manner in both NIH / 3T3 and D122 cells (FIGS. 2B and 2C). The antibody also strongly inhibited PDGF-BB-stimulated phosphorylation of both Akt and p44 / 42 MAP kinase in D122 cells (FIG. 2D).

Antitumor Activity of Anti- mPDGFR β Antibody in Tumor Xenograft Model . 3 ovarian carcinomas (SK-OV-3, OV-CAR-5 and OV-CAR-8), 1 pancreatic carcinoma (BxPC3), 1 lung carcinoma (NCI-H460) and 1 renal carcinoma (Caki-1 Six human xenograft tumor models including) were used to assess the antitumor activity of 1B3 in vivo. Athymic (nu / nu) mice (females, 7-8 weeks old) were housed for 7-10 days prior to xenograft transplantation. Each mouse was injected subcutaneously with 3-10 × 10 ⁶ cells of SK-OV-3, OVCAR-5, OVCAR-8, Caki-1, BxPC3 or NCI-H460 tumor cells. When tumors reached about 250-300 mm ³ , mice were randomly divided into groups of 10 to 12 animals each and treated by intraperitoneal injection 2 to 3 times weekly in combination with 1B3, DC101 or 1B3 in combination with DC101 It was. Human IgG and / or USP saline was used as a control. Tumor size and mouse weight were measured twice weekly. Tumor volume was calculated using the equation π / 6 x (length x width ² ), where length is the longest diameter and width is the diameter perpendicular to length. The ratio (T / C%) of the mean tumor volume of the treated group to the mean tumor volume of the control group was calculated. Statistical analysis was performed by Student's t-test.

To study the antitumor effects of monotherapy with anti-PDGFRβ antibodies, mice were treated by intraperitoneal injection twice weekly with 1B3, normal human IgG, or saline. In the SK-OV-3 model, tumor-bearing mice were dosed with 6, 20, or 60 mg / kg doses of 1B3. No tumor growth inhibition was observed in mice treated with two lower doses of 1B3 (FIG. 3A). At 60 mg / kg, 1B3 completely blocked tumor growth up to 29 days after initiation of treatment (P = 0.0028), after which tumors grew rapidly at rates similar to human IgG-treated controls despite continuous antibody treatment. Started (FIG. 3A). 1B3 (at 60 mg / kg dose) also showed significant antitumor activity in both OV-CAR-8 and NCI-H460 xenograft models: 1B3 almost completely prevented the growth of OV-CAR-8 tumors during the treatment. Blocked (FIG. 3B) (P = 0.0022) and significantly slowed the growth of NCI-H460 tumors (FIG. 3F) (P <0.05). In contrast, 1B3 (three times weekly at 40 mg / kg or twice weekly at 60 mg / kg) did not show any antitumor activity in BxPC3, OV-CAR-5 and Caki-1 xenograft models (FIG. 3C, 3D and 3E). 1B3 treatment did not cause any apparent toxicity, including changes in body weight and animal behavior in all six models tested.

Enhancement of antitumor and anti-angiogenic activity of DC101 , an anti- VEGFR2 antibody by 1B3, in a tumor xenograft model . Anti-tumor and anti-angiogenic activity of anti-VEGFR2 antibodies has been demonstrated in many animal models (Prewett, M. et al., 1999, Cancer Res. 59: 5209-18). In this example, two xenograft models, BxPC-3 (pancreatic carcinoma) and NCI-H460 (non-small cell lung carcinoma), can be used to enhance the antitumor and anti-angiogenic activity of DC101. Prove that. Athymic nude mice carrying xenograft tumors of 300-350 mm ³ size were randomly divided and divided into 4 treatment groups (n = 12 / group), saline, 1B3 (40 mg / kg), DC101 (40 mg / kg), or 1B3 (40 mg / kg) + DC101 (40 mg / kg) by intraperitoneal injection three times weekly. As expected, DC101 treatment significantly inhibited the growth of both BxPC3 and NCI-H460 xenografts (P = 0.0001 and <0.0001, respectively) (FIGS. 4A and 4B). Monotherapy with 1B3 showed moderate antitumor activity in the NCI-H460 model (T / C% = 66%, P = 0.0062 at 22 days after treatment) but did not show any antitumor activity in BxPC3 xenografts. The combination of 1B3 and DC101 significantly improved tumor suppression in the BxPC3 model, with T / C% at the end of the therapy being 27.3% in mice receiving the antibody combination, 38.7% in mice treated with DC101 alone. Compared (P = 0.0346). In addition, tumor regression was observed in 7 out of 12 mice (58.3%) in the antibody combination group, compared to 2 out of 11 mice (18.2%) in the DC101 alone treatment group and administered 1B3 alone. Degeneration was not observed in mice. In the NCI-H460 model, 1B3 + DC101 also showed much stronger tumor suppressive activity than 1B3 or DC101 alone, and at 22 days after treatment, T / C% was 22% in the 1B3 + DC101 group, which was in the 1B3 group. Compared with 66% (P <0.0001); At the end of the study (50 days post-treatment), the mean tumor volume in the antibody combination group was 2 times smaller than in the DC101 alone treatment group (813.9 ± 127.8 mm ³ versus 1660.9 ± 554.4 mm ³ ) (P = 0.025). There were no deaths in the antibody combination group, but two out of 12 mice died during the study in the DC101 group (one on day 22 and the other on day 43).

Immunohistochemistry of Tumor Sections . Tumor xenografts from antibody-treated mice were examined by immunohistochemical staining for both vascular density and perivascular cell coverage on blood vessels. At 34 days post-treatment (BxPC3 model) or 50 days (NCI-H460 model), 6 mice each were euthanized from the DC101 monotherapy group and the DC101 / 1B3 combination therapy group. Tumors were removed and fixed overnight at 4 ° C. in 10% neutral buffered formalin and paraffin embedded. Fixed tumors were made into 6 μm thick sections with a Leica RM2135 microtome and then double stained for both vascular and perivascular / SMC coverage.

Tumor sections were stained with both anti-CD31 antibody (for vascular staining) and anti-α-smooth muscle actin (α-SMA) antibody (for perivascular cell staining). Sections were first incubated with rat anti-mouse PECAM-1 (anti-CD31 antibody, Pharmingen, San Diego, Calif.) Overnight at 4 ° C., followed by a biotin-SP-labeled monkey anti-rat IgG antibody It was then incubated with streptavidin-Cy3 conjugate (all of Jackson Immunore Research). Sections were further stained with anti-α-SMA antibody-FITC conjugate (Sigma). Finally, sections were backstained for 5 minutes at RT with ToPro (Molecular Probe, Leiden, The Netherlands).

Immunofluorescence images (200 × magnification) were acquired using EZ-C1 2.20 software through a digital camera (Nikon Eclipse TE2000U) connected to a confocal microscope. Computer assisted morphological quantification was performed using Image Pro Plus software (MediaCybernatics, Silver Spring, MD) and analyzed from each section.

Five digital images of tumor periphery and tumor center from each section were taken for total CD31 ⁺ vessels, α-SMA ⁺ vessels, mature vessels (dual CD31 ⁺ and α-SMA ⁺ ), and dual CD31 ⁺ / for total CD31 ⁺ vessels. Counting was done for percentage of α-SMA ⁺ . Two different quantitative methods were used to measure the vascular distribution of individual tumor sections: vessel density (number of vessels / mm ³ ) and percentage of vessel area (% vessel area to total tumor area). Computer assisted morphological quantification was performed using Image Pro Plus software. For statistical analysis, the 2-way ANOVA followed by the Fisher LSD method was used for multiple comparisons (Sigma Start 3.1 Seastart Software, Inc.).

DC101 treatment significantly reduced CD31 ⁺ vascular density in BxPC3 tumor xenografts, mainly within the tumor center (FIG. 5A), but showed only a weak effect on α-SMA ⁺ vascular density (FIG. 5B). The addition of 1B3 to DC101 did not further reduce vascular distribution at the center of the tumor, but significantly increased the decrease in vascular density at the periphery of the tumor (FIG. 5A). 1B3 alone did not show a significant effect on overall tumor vascular density (FIG. 5A), but decreased α-SMA staining at the periphery of the tumor (FIG. 5B), which indicated the percentage of CD31 / α-SMA double positive (mature) vessels. Reduced (FIG. 5C). This indicates that PDGFRβ-positive perivascular cells were selectively inhibited in the tumor. Interestingly, in both DC101 alone and DC101 + 1B3 treated tumors, the decrease in CD31 ⁺ blood vessels correlated directly with the decrease in α-SMA ⁺ perivascular cell coverage in both tumor center and periphery (FIGS. 5A and B). ). As a result, the percentage of mature blood vessels at both the periphery and the center of the tumor, ie the ratio of double CD31 ⁺ / α-SMA ⁺ blood vessels to total CD31 ⁺ blood vessels, was increased from tumors treated with saline, DC101 alone, or DC101 + 1B3. It did not appear to be significantly different between (FIG. 5C). Similar phenomena were also observed in NCI-H460 tumor xenografts (FIG. 5D-F). Finally, almost the same observations were obtained (not shown) in both tumor xenografts when determining tumor vascular distribution using an alternative scoring method that measures the percentage of vascular area to total tumor area.

Selection of Human Anti- hPDGFR β Antibodies . The phage display library described above was screened for phage-Fab binding to human PDGFRβ. As described above for 1B3, the DNA sequences encoding the heavy and light chain genes of 2C5 (Table 1), the leading hPDGFRβ-binding clones, were amplified by PCR and containing human κ light chain constant region and human γ1 heavy chain constant region. Cloned into an expression vector. Expression vectors were transfected into NS0 myeloma cells and stable clones expressing 2C5 antibodies were selected.

As determined by BIAcore analysis, 2C5 binds to hPDGFRβ with similar binding properties as 1B3 binds to mPDGFRβ. Like 1B3, 2C5 is specific for PDGFRβ and does not bind hPDGFRα. However, 1B3 binds to murine PDGFRβ but not human PDGFRβ, whereas 2C5 binds to both proteins (FIG. 6). In one experiment, the IC ₅₀ value for 2C5 determined by surface plasmon resonance was 1.36 × ^{10 −11} M for hPDGFRβ and 6.05 × ^{10 −11} M for mPDGFRβ. IC ₅₀ for 1B3 binding to hPDGFRβ was 4.17 × ^{10 −11} M.

2C5 also blocks the binding of PDGF-BB to both human and mouse PDGFRβ as shown in FIG. 6. IC ₅₀ values were 0.55 nM for hPDGFRβ and 0.35 nM for mPDGFRβ.

2 C5 inhibits PDGF - BB -stimulated receptor phosphorylation and downstream signaling in cell based assays . The ability of 2C5 to block PDGFRβ phosphorylation was determined for human Caki-1 tumor cells (FIG. 7) and mouse NIH / 3T3 and D122 cell lines (FIG. 8). 2C5 blocked receptor phosphorylation even at low concentrations tested (IC ₅₀ << 1.8 μg / ml). 2C5 also strongly inhibited PDGF-BB-stimulated phosphorylation of Akt and p44 / 42 MAP kinases in Caki-1 cells (FIG. 7) and D122 cells (FIG. 8).

Antitumor Activity of Anti - PDGFR β Antibody in Tumor Xenograft Model . The five human xenograft tumor models described above (OVCAR-5 and OVCAR-8, BxPC3, NCI-H460, and Caki-1) were used to assess the antitumor activity of 2C5 in vivo. 2C5, which binds to and inhibits the human PDGFRβ receptor of the tumor and the mouse receptor of the vascular system, was compared to 1B3, which only binds to the mouse vascular system. Moderate reductions in tumor growth were observed in the SKOV-3, OVCAR-8, and NCI-H460 models, but not in the OVCAR-5, BxPC3 and Caki-1 models (FIG. 9; Table 2).

Wherein in the tumor xenograft model - PDGFR β (2 C5) / anti - mVEGFR 2 (DC101) the combination of antibodies anti-tumor activity. 2C5 binding to human and mouse PDGFRβ receptors (ie, tumors of human xenografts in murine hosts and PDGFRβ receptors on the vascular system) were also tested for inhibition of tumor growth in combination with antibodies specific for mouse VEGFR2 (DC101). Xenograft cell lines were BxPC-3, MIA-PaCa-2, Detroit-562, HCT-8, NCI-H460, NCI-H292, and HCT-116. Co-administration of 2C5 and DC101 resulted in significantly greater inhibition of tumor growth than the two antibodies alone (FIG. 10 and Table 3).

2 C5 inhibits PDGF - BB -stimulated cell migration in cell based migration assays . Two-chamber technique was used to determine the inhibitory capacity of 2C5 in cell migration using NCI-H460, OVCAR8, WS-I and U-118 cell lines. Collagen (100 μg / ml) was added to the upper chamber (100 μl) and the lower chamber (150 μl) and incubated at 37 ° C. for 1 hour. After washing the chamber twice with PBS, 100 μl of deficient cells (estimated 0.5-1 × 10 ⁶ / ml in serum-free medium) were added to the upper chamber and 150 μl of serum-free medium was added to the lower chamber. Then incubated at 37 ° C. for 4 hours. For PDGF-BB stimulation, PDGF-BB was added to the lower chamber at a final concentration of 100 ng / ml. In 2C5 wells, 2C5 was added to the upper chamber at a final concentration of 30 μg / ml. Unmoved cells in the upper chamber were scraped from the membrane. After washing three times with PBS, migrated cells on the membrane on the lower chamber side were fixed with 4% formalin and stained with Hoechst (4 μg / ml). The results were captured and cells counted under fluorescence microscopy. At a concentration of 30 μg / ml, 2C5 completely inhibited PDGF-BB-stimulated cell migration in all four cell lines used for analysis (FIG. 11).

Antitumor activity of anti- PDGFR β (2 C5 ) / chemo or anti- PDGFR β (2C5) / chemo / anti-mVEGFR 2 (DC101) antibody combinations in tumor xenograft models . Anti-PDGFRβ Antibody 2C5 was also tested for inhibition of tumor growth in combination with chemotherapy (gemcitabine) or chemotherapy (gemcitabine, paclitaxel or oxaliplatin) + anti-VEGFR2 antibody (DC101). Xenograft cell lines were NCI-H292, MIA-PaCa-2, NCI-H460 and GEO. Co-administration of 2C5 and gemcitabine inhibited tumor growth more than 2C5 or gemcitabine alone. Co-administration of 2C5, chemo and DC101 inhibited tumor growth more than the combination of chemo / DC101. (Figure 12 and Table 4).

Wherein in the tumor xenograft model - PDGFR β (2 C5) / anti - PDGFR α (specific 3 G3, or α mPDGFR specific IE10 in the hPDGFR α) antibody combination of antitumor activity. Anti-PDGFRβ Antibody 2C5 was also tested for inhibition of tumor growth in combination with anti-PDGFRα antibody. Xenograft cell lines were Caki-1, HPAC and U-118MG. Co-administration of 2C5 and 3G3 significantly inhibited tumor growth significantly compared to using both antibodies alone (FIG. 13 and Table 5).

Effect of 2 C5 Antibodies on PDGFR β, PDGF - BB and VEGF Expression . NCI-H460 cell suspension ( ⁵ × 10 ⁵ cells / mouse) was injected subcutaneously into female athymic mice. When tumors reached about 250 mm ³ , mice were randomized and divided into four treatment groups (n = 35 for USP saline, 2C5, DC101 and 2C5 + DC101, respectively). At -43 days, 7 days and 14 days, 6 animals from each group were sacrificed and their tumors harvested. ELISA assays were performed using tumor lysates to evaluate the levels of PDGFRβ, PDGF-BB, mVEGFR2, mVEGF, hVEGF, FGF and HIF-1α (FIG. 14).

2 C5 binds to domain 1 (D1), and domain 2 (D2) of the β PDGFR. Various chimeric constructs of PDGFRβ and PDGFRα were first prepared by domain swapping starting from the N-terminus to the C-terminus, with the PDGFRβ domain (s) replacing the PDGFRα domain (s). PDGFRβ / α chimeras were tested for 2C5 binding capacity. Shorter versions of PDGFRβ were then prepared, including D1D2, D2D3 and D1D2D3, to identify 2C5 binding domains on PDGFRβ. The results show that 2C5 binds to PDGFRβ through both D1 and D2 of the receptor (FIG. 15 and Table 6).

                         SEQUENCE LISTING <110> IMCLONE LLC        ZHU, Zhenping        SHEN, Juqun   <120> PDGFR-beta-Specific Inhibitors <130> 11245/55301 <160> 46 <170> PatentIn version 3.3 <210> 1 <211> 354 <212> DNA <213> Homo sapiens <220> <221> CDS (222) (1) .. (354) <400> 1 cag gtg cag ctg cag gag tcg ggg gga ggc ctg gtc aag cct ggg ggg 48 Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly 1 5 10 15 tcc ctg aga ctc tcc tgt gca gcc tct gga ttc acc ttc agt agc tat 96 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr             20 25 30 agc atg aac tgg gtc cgc cag gct cca ggg aag ggg ctg gag tgg gtc 144 Ser Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val         35 40 45 tca tcc att agt agt agt agt agt tac ata tac tac gca gac tcc gtg 192 Ser Ser Ile Ser Ser Ser Ser Ser Tyr Ile Tyr Tyr Ala Asp Ser Val     50 55 60 aag ggc cgg ttc acc atc tcc aga gac aat tcc aag aac acg ctg tat 240 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 ctg caa atg aac agc ctg aga gcc gag gac acg gcc gtg tat tac tgt 288 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys                 85 90 95 gcg aaa ggg ggg cgc ccg ctc cta gtc ttt gac ttc tgg ggc cag gga 336 Ala Lys Gly Gly Arg Pro Leu Leu Val Phe Asp Phe Trp Gly Gln Gly             100 105 110 acc ctg gtc acc gtc tca 354 Thr Leu Val Thr Val Ser         115 <210> 2 <211> 118 <212> PRT <213> Homo sapiens <400> 2 Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr             20 25 30 Ser Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val         35 40 45 Ser Ser Ile Ser Ser Ser Ser Ser Tyr Ile Tyr Tyr Ala Asp Ser Val     50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys                 85 90 95 Ala Lys Gly Gly Arg Pro Leu Leu Val Phe Asp Phe Trp Gly Gln Gly             100 105 110 Thr Leu Val Thr Val Ser         115 <210> 3 <211> 15 <212> DNA <213> Homo sapiens <220> <221> CDS (222) (1) .. (15) <400> 3 agc tat agc atg aac 15 Ser Tyr Ser Met Asn 1 5 <210> 4 <211> 5 <212> PRT <213> Homo sapiens <400> 4 Ser Tyr Ser Met Asn 1 5 <210> 5 <211> 51 <212> DNA <213> Homo sapiens <220> <221> CDS (222) (1) .. (51) <400> 5 tcc att agt agt agt agt agt tac ata tac tac gca gac tcc gtg aag 48 Ser Ile Ser Ser Ser Ser Ser Tyr Ile Tyr Tyr Ala Asp Ser Val Lys 1 5 10 15 ggc 51 Gly                                                                          <210> 6 <211> 17 <212> PRT <213> Homo sapiens <400> 6 Ser Ile Ser Ser Ser Ser Ser Tyr Ile Tyr Tyr Ala Asp Ser Val Lys 1 5 10 15 Gly      <210> 7 <211> 30 <212> DNA <213> Homo sapiens <220> <221> CDS (222) (1) .. (30) <400> 7 ggg ggg cgc ccg ctc cta gtc ttt gac ttc 30 Gly Gly Arg Pro Leu Leu Val Phe Asp Phe 1 5 10 <210> 8 <211> 10 <212> PRT <213> Homo sapiens <400> 8 Gly Gly Arg Pro Leu Leu Val Phe Asp Phe 1 5 10 <210> 9 <211> 330 <212> DNA <213> Homo sapiens <220> <221> CDS (222) (1) .. (330) <400> 9 gaa att gtg atg aca cag tct cca ggc acc ctg tct ttg tct cca ggg 48 Glu Ile Val Met Thr Gln Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly 1 5 10 15 gaa aga gcc acc ctc tcc tgc agg gcc agt cag agt gtt agc agc agc 96 Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Ser             20 25 30 tac tta gcc tgg tac cag cag aaa cct ggc cag gct ccc agg ctc ctc 144 Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu         35 40 45 atc tat gat gca tcc aag agg gcc acc ggc atc cca gcc agg ttc agt 192 Ile Tyr Asp Ala Ser Lys Arg Ala Thr Gly Ile Pro Ala Arg Phe Ser     50 55 60 ggc agt ggg tct ggg aca gac ttc act ctc acc atc agc acc cta gag 240 Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Thr Leu Glu 65 70 75 80 tct gaa gat tct gca gtt tat tac tgt cag caa cgt ggc tac tgg cct 288 Ser Glu Asp Ser Ala Val Tyr Tyr Cys Gln Gln Arg Gly Tyr Trp Pro                 85 90 95 ccc atc acc ttc ggc caa ggg aca cga ctg gag att aaa cga 330 Pro Ile Thr Phe Gly Gln Gly Thr Arg Leu Glu Ile Lys Arg             100 105 110 <210> 10 <211> 110 <212> PRT <213> Homo sapiens <400> 10 Glu Ile Val Met Thr Gln Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly 1 5 10 15 Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Ser             20 25 30 Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu         35 40 45 Ile Tyr Asp Ala Ser Lys Arg Ala Thr Gly Ile Pro Ala Arg Phe Ser     50 55 60 Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Thr Leu Glu 65 70 75 80 Ser Glu Asp Ser Ala Val Tyr Tyr Cys Gln Gln Arg Gly Tyr Trp Pro                 85 90 95 Pro Ile Thr Phe Gly Gln Gly Thr Arg Leu Glu Ile Lys Arg             100 105 110 <210> 11 <211> 36 <212> DNA <213> Homo sapiens <220> <221> CDS (222) (1) .. (36) <400> 11 agg gcc agt cag agt gtt agc agc agc tac tta gcc 36 Arg Ala Ser Gln Ser Val Ser Ser Ser Tyr Leu Ala 1 5 10 <210> 12 <211> 12 <212> PRT <213> Homo sapiens <400> 12 Arg Ala Ser Gln Ser Val Ser Ser Ser Tyr Leu Ala 1 5 10 <210> 13 <211> 21 <212> DNA <213> Homo sapiens <220> <221> CDS (222) (1) .. (21) <400> 13 gat gca tcc aag agg gcc acc 21 Asp Ala Ser Lys Arg Ala Thr 1 5 <210> 14 <211> 7 <212> PRT <213> Homo sapiens <400> 14 Asp Ala Ser Lys Arg Ala Thr 1 5 <210> 15 <211> 30 <212> DNA <213> Homo sapiens <220> <221> CDS (222) (1) .. (30) <400> 15 cag caa cgt ggc tac tgg cct ccc atc acc 30 Gln Gln Arg Gly Tyr Trp Pro Pro Ile Thr 1 5 10 <210> 16 <211> 10 <212> PRT <213> Homo sapiens <400> 16 Gln Gln Arg Gly Tyr Trp Pro Pro Ile Thr 1 5 10 <210> 17 <211> 360 <212> DNA <213> Homo sapiens <220> <221> CDS (222) (1) .. (360) <400> 17 cag gtg cag ctg gtg cag tct ggg gct gag gtg aag aag cct ggg tcc 48 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser 1 5 10 15 tcg gtg aag gtc tcc tgc aag gct tct gga ggc acc ttc agc agc tat 96 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Gly Thr Phe Ser Ser Tyr             20 25 30 gct atc agc tgg gtg cga cag gcc cct gga caa ggg ctt gag tgg atg 144 Ala Ile Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met         35 40 45 gga agg atc atc cct atc ctt ggt ata gca aac tac gca cag aag ttc 192 Gly Arg Ile Ile Pro Ile Leu Gly Ile Ala Asn Tyr Ala Gln Lys Phe     50 55 60 cag ggc aga gtc acg att acc gcg gac aaa tcc acg agc aca gcc tac 240 Gln Gly Arg Val Thr Ile Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr 65 70 75 80 atg gag ctg agc agc ctg aga tct gag gac acg gcc gtc tat tac tgt 288 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys                 85 90 95 gcg aga gat atg ggt tca agg aat tat tat tac ttc tac tgg ggc cag 336 Ala Arg Asp Met Gly Ser Arg Asn Tyr Tyr Tyr Phe Tyr Trp Gly Gln             100 105 110 gga acc ctg gtc acc gtc tca agc 360 Gly Thr Leu Val Thr Val Ser Ser         115 120 <210> 18 <211> 120 <212> PRT <213> Homo sapiens <400> 18 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Gly Thr Phe Ser Ser Tyr             20 25 30 Ala Ile Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met         35 40 45 Gly Arg Ile Ile Pro Ile Leu Gly Ile Ala Asn Tyr Ala Gln Lys Phe     50 55 60 Gln Gly Arg Val Thr Ile Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys                 85 90 95 Ala Arg Asp Met Gly Ser Arg Asn Tyr Tyr Tyr Phe Tyr Trp Gly Gln             100 105 110 Gly Thr Leu Val Thr Val Ser Ser         115 120 <210> 19 <211> 15 <212> DNA <213> Homo sapiens <220> <221> CDS (222) (1) .. (15) <400> 19 agc tat gct atc agc 15 Ser Tyr Ala Ile Ser 1 5 <210> 20 <211> 5 <212> PRT <213> Homo sapiens <400> 20 Ser Tyr Ala Ile Ser 1 5 <210> 21 <211> 51 <212> DNA <213> Homo sapiens <220> <221> CDS (222) (1) .. (51) <400> 21 agg atc atc cct atc ctt ggt ata gca aac tac gca cag aag ttc cag 48 Arg Ile Ile Pro Ile Leu Gly Ile Ala Asn Tyr Ala Gln Lys Phe Gln 1 5 10 15 ggc 51 Gly                                                                          <210> 22 <211> 17 <212> PRT <213> Homo sapiens <400> 22 Arg Ile Ile Pro Ile Leu Gly Ile Ala Asn Tyr Ala Gln Lys Phe Gln 1 5 10 15 Gly      <210> 23 <211> 33 <212> DNA <213> Homo sapiens <220> <221> CDS (222) (1) .. (33) <400> 23 gat atg ggt tca agg aat tat tat tac ttc tac 33 Asp Met Gly Ser Arg Asn Tyr Tyr Tyr Phe Tyr 1 5 10 <210> 24 <211> 11 <212> PRT <213> Homo sapiens <400> 24 Asp Met Gly Ser Arg Asn Tyr Tyr Tyr Phe Tyr 1 5 10 <210> 25 <211> 324 <212> DNA <213> Homo sapiens <220> <221> CDS (222) (1) .. (324) <400> 25 gaa att gtg ctg act cag tct cca gcc acc ctg tct ttg tct cca ggg 48 Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly 1 5 10 15 gaa aga gcc acc ctc tcc tgc agg gcc agt cag agt gtt ggc agg tac 96 Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Gly Arg Tyr             20 25 30 tta gcc tgg tac caa cag aaa cct ggc cag gct ccc agg ctc ctc atc 144 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile         35 40 45 tat ggt gca tcc aac agg gcc act ggc atc cca gcc agg ttc agt ggc 192 Tyr Gly Ala Ser Asn Arg Ala Thr Gly Ile Pro Ala Arg Phe Ser Gly     50 55 60 agt ggg tct ggg aca gac ttc act ctc acc atc agc agc cta gag cct 240 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro 65 70 75 80 gaa gat ttt gca gtt tat tac tgt cag cag cgt agc aac tgg cct ctc 288 Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Arg Ser Asn Trp Pro Leu                 85 90 95 act ttc ggc gga ggg acc aag gtg gag atc aaa cga 324 Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Arg             100 105 <210> 26 <211> 108 <212> PRT <213> Homo sapiens <400> 26 Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly 1 5 10 15 Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Gly Arg Tyr             20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile         35 40 45 Tyr Gly Ala Ser Asn Arg Ala Thr Gly Ile Pro Ala Arg Phe Ser Gly     50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro 65 70 75 80 Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Arg Ser Asn Trp Pro Leu                 85 90 95 Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Arg             100 105 <210> 27 <211> 33 <212> DNA <213> Homo sapiens <220> <221> CDS (222) (1) .. (33) <400> 27 agg gcc agt cag agt gtt ggc agg tac tta gcc 33 Arg Ala Ser Gln Ser Val Gly Arg Tyr Leu Ala 1 5 10 <210> 28 <211> 11 <212> PRT <213> Homo sapiens <400> 28 Arg Ala Ser Gln Ser Val Gly Arg Tyr Leu Ala 1 5 10 <210> 29 <211> 21 <212> DNA <213> Homo sapiens <220> <221> CDS (222) (1) .. (21) <400> 29 ggt gca tcc aac agg gcc act 21 Gly Ala Ser Asn Arg Ala Thr 1 5 <210> 30 <211> 7 <212> PRT <213> Homo sapiens <400> 30 Gly Ala Ser Asn Arg Ala Thr 1 5 <210> 31 <211> 27 <212> DNA <213> Homo sapiens <220> <221> CDS (222) (1) .. (27) <400> 31 cag cag cgt agc aac tgg cct ctc act 27 Gln Gln Arg Ser Asn Trp Pro Leu Thr 1 5 <210> 32 <211> 9 <212> PRT <213> Homo sapiens <400> 32 Gln Gln Arg Ser Asn Trp Pro Leu Thr 1 5 <210> 33 <211> 348 <212> DNA <213> Homo sapiens <220> <221> CDS (222) (1) .. (348) <400> 33 gag gtg cag ctg gtg cag tct ggg gga ggc ctg gtc aag cct ggg ggg 48 Glu Val Gln Leu Val Gln Ser Gly Gly Gly Leu Val Lys Pro Gly Gly 1 5 10 15 tcc ctg aga ctc tcc tgt gca gcc tct gga ttc acc ttc agt agc tat 96 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr             20 25 30 agc atg aac tgg gtc cgc cag gct cca ggg aag ggg ctg gag tgg gtc 144 Ser Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val         35 40 45 tca tcc att agt agt agt agt agt tac ata tac tac gca gac tca gtg 192 Ser Ser Ile Ser Ser Ser Ser Ser Tyr Ile Tyr Tyr Ala Asp Ser Val     50 55 60 aag ggc cga ttc acc atc tcc aga gac aac gcc aag aac tca ctg tat 240 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr 65 70 75 80 ctg caa atg aac agc ctg aga gcc gag gac acg gct gtg tat tac tgt 288 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys                 85 90 95 gcg aga gtc aca gat gct ttt gat atc tgg ggc caa ggg aca atg gtc 336 Ala Arg Val Thr Asp Ala Phe Asp Ile Trp Gly Gln Gly Thr Met Val             100 105 110 acc gtc tca agc 348 Thr Val Ser Ser         115 <210> 34 <211> 116 <212> PRT <213> Homo sapiens <400> 34 Glu Val Gln Leu Val Gln Ser Gly Gly Gly Leu Val Lys Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr             20 25 30 Ser Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val         35 40 45 Ser Ser Ile Ser Ser Ser Ser Ser Tyr Ile Tyr Tyr Ala Asp Ser Val     50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys                 85 90 95 Ala Arg Val Thr Asp Ala Phe Asp Ile Trp Gly Gln Gly Thr Met Val             100 105 110 Thr Val Ser Ser         115 <210> 35 <211> 321 <212> DNA <213> Homo sapiens <220> <221> CDS (222) (1) .. (321) <400> 35 gaa att gtg atg aca cag tct cca gcc acc ctg tct ttg tct cca ggg 48 Glu Ile Val Met Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly 1 5 10 15 gaa aga gcc acc ctc tcc tgc agg gcc agt cag agt gtt agc agc tac 96 Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Tyr             20 25 30 tta gcc tgg tac caa cag aaa cct ggc cag gct ccc agg ctc ctc atc 144 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile         35 40 45 tat gat tca tcc aac agg gcc act ggc atc cca gcc aga ttc agt ggc 192 Tyr Asp Ser Ser Asn Arg Ala Thr Gly Ile Pro Ala Arg Phe Ser Gly     50 55 60 agt ggg tct ggg aca gac ttc act ctc acc atc agc agc cta gag cct 240 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro 65 70 75 80 gaa gat ttt gca act tat tac tgt cta cag cat aac act ttt cct ccg 288 Glu Asp Phe Ala Thr Tyr Tyr Cys Leu Gln His Asn Thr Phe Pro Pro                 85 90 95 acg ttc ggc caa ggg acc aag gtg gaa atc aaa 321 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys             100 105 <210> 36 <211> 107 <212> PRT <213> Homo sapiens <400> 36 Glu Ile Val Met Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly 1 5 10 15 Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Tyr             20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile         35 40 45 Tyr Asp Ser Ser Asn Arg Ala Thr Gly Ile Pro Ala Arg Phe Ser Gly     50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Leu Gln His Asn Thr Phe Pro Pro                 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys             100 105 <210> 37 <211> 321 <212> DNA <213> Homo sapiens <220> <221> CDS (222) (1) .. (321) <400> 37 gac atc cag atg acc cag tct cca tct tcc gtg tct gca tct ata gga 48 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Val Ser Ala Ser Ile Gly 1 5 10 15 gac aga gtc acc atc act tgt cgg gcg agt cag ggt att gac aac tgg 96 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Gly Ile Asp Asn Trp             20 25 30 tta ggc tgg tat cag cag aaa cct ggg aaa gcc cct aaa ctc ctg atc 144 Leu Gly Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile         35 40 45 tac gat gca tcc aat ttg gac aca ggg gtc cca tca agg ttc agt gga 192 Tyr Asp Ala Ser Asn Leu Asp Thr Gly Val Pro Ser Arg Phe Ser Gly     50 55 60 agt gga tct ggg aca tat ttt act ctc acc atc agt agc ctg caa gct 240 Ser Gly Ser Gly Thr Tyr Phe Thr Leu Thr Ile Ser Ser Leu Gln Ala 65 70 75 80 gaa gat ttt gca gtt tat ttc tgt caa cag gct aaa gct ttt cct ccc 288 Glu Asp Phe Ala Val Tyr Phe Cys Gln Gln Ala Lys Ala Phe Pro Pro                 85 90 95 act ttc ggc gga ggg acc aag gtg gac atc aaa 321 Thr Phe Gly Gly Gly Thr Lys Val Asp Ile Lys             100 105 <210> 38 <211> 107 <212> PRT <213> Homo sapiens <400> 38 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Val Ser Ala Ser Ile Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Gly Ile Asp Asn Trp             20 25 30 Leu Gly Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile         35 40 45 Tyr Asp Ala Ser Asn Leu Asp Thr Gly Val Pro Ser Arg Phe Ser Gly     50 55 60 Ser Gly Ser Gly Thr Tyr Phe Thr Leu Thr Ile Ser Ser Leu Gln Ala 65 70 75 80 Glu Asp Phe Ala Val Tyr Phe Cys Gln Gln Ala Lys Ala Phe Pro Pro                 85 90 95 Thr Phe Gly Gly Gly Thr Lys Val Asp Ile Lys             100 105 <210> 39 <211> 348 <212> DNA <213> Mus musculus <220> <221> CDS (222) (1) .. (348) <400> 39 cag gtc aaa ctg cag cag tct ggg gca gag ctt gtc aag cca ggg gcc 48 Gln Val Lys Leu Gln Gln Ser Gly Ala Glu Leu Val Lys Pro Gly Ala 1 5 10 15 tca gtc aag ttg tcc tgc aca gct tct ggc ttc aac att aaa gac acc 96 Ser Val Lys Leu Ser Cys Thr Ala Ser Gly Phe Asn Ile Lys Asp Thr             20 25 30 tat ata cac tgg gtg aag cag agc cct gaa cag ggc ctg gag tgg att 144 Tyr Ile His Trp Val Lys Gln Ser Pro Glu Gln Gly Leu Glu Trp Ile         35 40 45 gga agg atc gat cct ccg aat gat aat act aaa tat gac ccg aag ttc 192 Gly Arg Ile Asp Pro Pro Asn Asp Asn Thr Lys Tyr Asp Pro Lys Phe     50 55 60 cag ggc aag gcc act ata aca gca gac aca tcc tcc aat aca gcc tac 240 Gln Gly Lys Ala Thr Ile Thr Ala Asp Thr Ser Ser Asn Thr Ala Tyr 65 70 75 80 atg cag ctc cgc agc ctg aca tct gag gac act gcc gtc tat tac tgt 288 Met Gln Leu Arg Ser Leu Thr Ser Glu Asp Thr Ala Val Tyr Tyr Cys                 85 90 95 gcc ctc cca ccg ttc tac ttt gac tac tgg ggc cat ggc acc acg gtc 336 Ala Leu Pro Pro Phe Tyr Phe Asp Tyr Trp Gly His Gly Thr Thr Val             100 105 110 acc gtc tcc tca 348 Thr Val Ser Ser         115 <210> 40 <211> 116 <212> PRT <213> Mus musculus <400> 40 Gln Val Lys Leu Gln Gln Ser Gly Ala Glu Leu Val Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Leu Ser Cys Thr Ala Ser Gly Phe Asn Ile Lys Asp Thr             20 25 30 Tyr Ile His Trp Val Lys Gln Ser Pro Glu Gln Gly Leu Glu Trp Ile         35 40 45 Gly Arg Ile Asp Pro Pro Asn Asp Asn Thr Lys Tyr Asp Pro Lys Phe     50 55 60 Gln Gly Lys Ala Thr Ile Thr Ala Asp Thr Ser Ser Asn Thr Ala Tyr 65 70 75 80 Met Gln Leu Arg Ser Leu Thr Ser Glu Asp Thr Ala Val Tyr Tyr Cys                 85 90 95 Ala Leu Pro Pro Phe Tyr Phe Asp Tyr Trp Gly His Gly Thr Thr Val             100 105 110 Thr Val Ser Ser         115 <210> 41 <211> 327 <212> DNA <213> Mus musculus <220> <221> CDS (222) (1) .. (327) <400> 41 gac atc gag ctc act cag tct cca aaa ttc atg tcc aca tca gta gga 48 Asp Ile Glu Leu Thr Gln Ser Pro Lys Phe Met Ser Thr Ser Val Gly 1 5 10 15 gac agg gtc agc gtc acc tgc aag gcc agt cag aat gtg gat act aat 96 Asp Arg Val Ser Val Thr Cys Lys Ala Ser Gln Asn Val Asp Thr Asn             20 25 30 gta gcc tgg tat caa cag aaa cca ggg caa tct cct aaa gca ctg att 144 Val Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ser Pro Lys Ala Leu Ile         35 40 45 tac tcg gca tcc tac cgg tac agt gga gtc cct gat cgc ttc aca ggc 192 Tyr Ser Ala Ser Tyr Arg Tyr Ser Gly Val Pro Asp Arg Phe Thr Gly     50 55 60 agt gga tct ggg aca gat ttc act ctc acc atc agc aat gtg cag tct 240 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Asn Val Gln Ser 65 70 75 80 gaa gac ttg gca gag tat ttc tgt cag caa tat aac agc ttt cct tac 288 Glu Asp Leu Ala Glu Tyr Phe Cys Gln Gln Tyr Asn Ser Phe Pro Tyr                 85 90 95 acg ttc gga ggg ggg acc aag ctg gaa ata aaa cgg gcg 327 Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg Ala             100 105 <210> 42 <211> 109 <212> PRT <213> Mus musculus <400> 42 Asp Ile Glu Leu Thr Gln Ser Pro Lys Phe Met Ser Thr Ser Val Gly 1 5 10 15 Asp Arg Val Ser Val Thr Cys Lys Ala Ser Gln Asn Val Asp Thr Asn             20 25 30 Val Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ser Pro Lys Ala Leu Ile         35 40 45 Tyr Ser Ala Ser Tyr Arg Tyr Ser Gly Val Pro Asp Arg Phe Thr Gly     50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Asn Val Gln Ser 65 70 75 80 Glu Asp Leu Ala Glu Tyr Phe Cys Gln Gln Tyr Asn Ser Phe Pro Tyr                 85 90 95 Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg Ala             100 105 <210> 43 <211> 378 <212> DNA <213> Homo sapiens <220> <221> CDS (222) (1) .. (378) <400> 43 cag gcg cag gtg gtg gag tct ggg gga ggc gtg gtc cag tct ggg agg 48 Gln Ala Gln Val Val Glu Ser Gly Gly Gly Val Val Gln Ser Gly Arg 1 5 10 15 tcc ctg aga ctc tcc tgt gca gcg tct gga ttc gcc ttc agt agc tac 96 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Ala Phe Ser Ser Tyr             20 25 30 ggc atg cac tgg gtc cgc cag gct cca ggc aag ggg ctg gag tgg gtg 144 Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val         35 40 45 gca gtt ata tgg tat gat gga agt aat aaa tac tat gca gac tcc gtg 192 Ala Val Ile Trp Tyr Asp Gly Ser Asn Lys Tyr Tyr Ala Asp Ser Val     50 55 60 agg ggc cga ttc acc atc tcc aga gac aat tcc gag aac acg ctg tat 240 Arg Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Glu Asn Thr Leu Tyr 65 70 75 80 ctg caa atg aac agc ctg aga gcc gag gac acc gct gtg tat tac tgt 288 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys                 85 90 95 gcc aga gat cac tat ggt tcg ggg gtg cac cac tat ttc tac tac ggt 336 Ala Arg Asp His Tyr Gly Ser Gly Val His His Tyr Phe Tyr Tyr Gly             100 105 110 ctg gac gtc tgg ggc caa ggg acc acg gtc acc gtc tcc tca 378 Leu Asp Val Trp Gly Gln Gly Thr Thr Val Val Val Ser Ser         115 120 125 <210> 44 <211> 126 <212> PRT <213> Homo sapiens <400> 44 Gln Ala Gln Val Val Glu Ser Gly Gly Gly Val Val Gln Ser Gly Arg 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Ala Phe Ser Ser Tyr             20 25 30 Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val         35 40 45 Ala Val Ile Trp Tyr Asp Gly Ser Asn Lys Tyr Tyr Ala Asp Ser Val     50 55 60 Arg Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Glu Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys                 85 90 95 Ala Arg Asp His Tyr Gly Ser Gly Val His His Tyr Phe Tyr Tyr Gly             100 105 110 Leu Asp Val Trp Gly Gln Gly Thr Thr Val Val Val Ser Ser         115 120 125 <210> 45 <211> 324 <212> DNA <213> Homo sapiens <220> <221> CDS (222) (1) .. (324) <400> 45 gaa att gtg ttg acg cag tct cca ggc acc ctg tct ttg tct cca ggg 48 Glu Ile Val Leu Thr Gln Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly 1 5 10 15 gaa aga gcc acc ctc tcc tgc agg gcc agt cag agt gtt agc agc agc 96 Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Ser             20 25 30 tac tta gcc tgg tac cag cag aaa cct ggc cag gct ccc agg ctc ctc 144 Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu         35 40 45 atc tat ggt gca tcc agc agg gcc act ggc atc cca gac agg ttc agt 192 Ile Tyr Gly Ala Ser Ser Arg Ala Thr Gly Ile Pro Asp Arg Phe Ser     50 55 60 ggc agt ggg tct ggg aca gac ttc act ctc acc atc agc aga ctg gag 240 Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Arg Leu Glu 65 70 75 80 cct gaa gat ttt gca gtg tat tac tgt cag cag tat ggt agc tca ccg 288 Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Tyr Gly Ser Ser Pro                 85 90 95 ctc act ttc ggc gga ggg acc aag gtg gag atc aaa 324 Leu Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys             100 105 <210> 46 <211> 108 <212> PRT <213> Homo sapiens <400> 46 Glu Ile Val Leu Thr Gln Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly 1 5 10 15 Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Ser             20 25 30 Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu         35 40 45 Ile Tyr Gly Ala Ser Ser Arg Ala Thr Gly Ile Pro Asp Arg Phe Ser     50 55 60 Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Arg Leu Glu 65 70 75 80 Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Tyr Gly Ser Ser Pro                 85 90 95 Leu Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys             100 105  

Claims (32)

An isolated antibody or fragment thereof that binds to domain 2 or domains 1 and 2 of platelet derived growth factor receptor-β (PDGFRβ). The composition of claim 1, wherein SEQ ID NO: 1 relative to SEQ ID NO: 20 in CDRH1; For SEQ ID NO: 22 in CDRH2; For SEQ ID NO: 24 in CDRH3; For SEQ ID NO: 28 in CDRL1; For SEQ ID NO: 30 in CDRL2; And an antibody or fragment thereof comprising a complementarity determining region that is at least about 90% homologous to SEQ ID NO: 32 in CDRL3. The antibody of claim 2 comprising the heavy chain variable domain amino acid sequence of SEQ ID NO: 18 and the light chain variable domain amino acid sequence of SEQ ID NO: 26. For SEQ ID NO: 4 in CDRH1; For SEQ ID NO: 6 in CDRH2; For SEQ ID NO: 8 in CDRH3; For SEQ ID NO: 12 in CDRL1; For SEQ ID NO: 14 in CDRL2; And an antibody or fragment thereof that specifically binds to platelet derived growth factor receptor-β (PDGFRβ) comprising a complementarity determining region that is at least about 90% homologous to SEQ ID NO: 16 in CDRL3. The antibody of claim 4 comprising the heavy chain variable domain amino acid sequence of SEQ ID NO: 2 and the light chain variable domain amino acid sequence of SEQ ID NO: 10. The antibody of claim 1, wherein the antibody is selected from the group consisting of single chain antibodies, Fabs, single chain Fv, diabodies, triabodies, and single domain antibodies. The antibody of any one of claims 1-5, which is a bispecific antibody. 8. The antibody of claim 7, which binds to a VEGFR receptor. The antibody of claim 8, wherein the VEGFR receptor is selected from the group consisting of VEGFR1, VEGFR2 and VEGFR3. An isolated polynucleotide encoding an amino acid sequence selected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 10, SEQ ID NO: 18, and SEQ ID NO: 26. The isolated polynucleotide of claim 10, comprising a nucleotide sequence that is at least 70% identical to a nucleotide sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 9, SEQ ID NO: 17, and SEQ ID NO: 25. An isolated polynucleotide that hybridizes to the complement of a nucleic acid molecule comprising a nucleotide sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 9, SEQ ID NO: 17, and SEQ ID NO: 25 under stringent conditions. An expression vector comprising said polynucleotide sequence operably linked to a regulatory control sequence that affects the expression of a polynucleotide according to claim 10 in a host cell. Recombinant host cell comprising the expression vector according to claim 13. The recombinant host cell of claim 14, wherein the recombinant host cell expresses an antibody that binds PDGFRβ. A method for producing an antibody that binds PDGFRβ, comprising culturing the cell according to claim 15 under conditions that allow expression of the antibody. A method for modulating the activity of PDGFRβ in a mammal, comprising administering to the mammal an effective amount of the antibody according to any one of claims 1 to 9. A method of inhibiting angiogenesis in a mammal, comprising administering to the mammal an effective amount of the antibody of any one of claims 1 to 9. A method of reducing tumor growth in a mammal comprising administering to the mammal an effective amount of the antibody according to any one of claims 1 to 9. 20. The method of claim 19, further comprising administering an effective amount of an anti-neoplastic agent or radiation. A method of inhibiting angiogenesis or reducing tumor growth in a mammal comprising administering to the mammal an effective amount of a specific antagonist of platelet derived growth factor-β (PDGFRβ) and an effective amount of a VEGFR antagonist. The method of claim 21, wherein said PDGFRβ-specific antagonist is an antibody that binds PDGFRβ. The method of claim 22, wherein said antibody blocks PDGFRβ dimerization. The method of claim 22, wherein the antibody binds to domain 2 or domains 1 and 2 of PDGFRβ. The antibody of claim 24, wherein the antibody is directed against SEQ ID NO: 20 in CDRH1; For SEQ ID NO: 22 in CDRH2; For SEQ ID NO: 24 in CDRH3; For SEQ ID NO: 28 in CDRL1; For SEQ ID NO: 30 in CDRL2; And a complementarity determining region that is at least about 90% homologous to SEQ ID NO: 32 in CDRL3. The method of claim 25, wherein the antibody comprises the heavy chain variable domain amino acid sequence of SEQ ID NO: 18 and the light chain variable domain amino acid sequence of SEQ ID NO: 26. The method of claim 21, wherein said PDGFRβ-specific antagonist is a bispecific antibody that binds PDGFRβ and a VEGF receptor selected from the group consisting of VEGFR1, VEGFR2, and VEGFR3. The method of claim 27, wherein the VEGF antagonist is an antibody that binds VEGFR2. The method of claim 21, wherein the PDGFRβ-specific antagonist is a bispecific antibody that binds PDGFRβ and VEGFR ligands. The method of claim 29, wherein the VEGFR ligand is selected from the group consisting of VEGF and PlGF. The method of claim 21, wherein the VEGFR antagonist is a soluble fragment of the VEGF receptor. The method of claim 21, further comprising administering an effective amount of an anti-neoplastic agent or radiation.

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